New clone of newcastle disease virus, its manufacture and its application in the medical treatment of cancer

ABSTRACT

A new clone of Newcastle disease virus which is interferon insensitive and has an ICPI between 1.2 and 2.0 and which may be used in the treatment of cancer and other diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 12/806,494, filedAug. 13, 2010 and is a non-provisional patent application based uponU.S. provisional application Ser. No. 61/283,154 filed Nov. 30, 2009.

BACKGROUND OF THE INVENTION

The information in the ASCII text file named 740481-54CONSeqListing.txt,created Jan. 14, 2013 and having a size of 25.8 kb is herebyincorporated by reference.

Newcastle Disease Virus (NDV) is a virus well known in the art [Diseasesof poultry, 10th edition, edited by B. W. Calnek, Mosby International,Iowa State University Press, Ames, Iowa, 1997]. The virus is responsiblefor great economic losses in the poultry industry. It is also well knownthat many strains of NDV exist (EP 0770397 B1) with an enormous range inthe severity and type of disease produced in poultry. Newcastle diseasevirus (NDV) is classified as an avian paramyxovirus-1 (APMV1), a memberof the family Paramyxoviridae in the order Mononegavirales. Members ofthis family have a single stranded, linear, RNA, with an ellipticalsymmetry. The total genome is roughly 16,000 nucleotides. Replication ofthe virus takes place in the cytoplasm of the host cell.

This family is divided into two subfamilies, the Paramyxovirinae and thePneumovirinae. During 1993, the International Committee on the Taxonomyof Viruses rearranged the paramyxoviruses and placed NDV within theRubulavirus genus. The genomes of most rubulaviruses, except NDV,contain a small hydrophobic (SH) protein gene that is not present amongother paramyxoviruses. Based on predicted amino acid sequences for eachviral protein, NDV clones phylogenetically group as a clade separatefrom the rubulaviruses. The polycistronic phosphoprotein (P) geneediting sequence of NDV and putative gene products are more similar toexpression patterns among members of the Respirovirus and Morbillivirus.In addition, structure of the nucleocapsid protein more closelyresembles the Respiroviruses. There are nine recognized serotypes amongavian paramyxoviruses that infect primarily only bird species. Thesevirus types are phylogenetically distinct from NDV, but separate as aclade with NDV from the other paramyxoviruses. This relationship wasfurther confirmed by phylogenetic analysis of full-length genomicsequences.

As with the situation for many other avian viruses, NDV has evolvedamong birds separate from their mammalian counterparts. Consequently,based on several key factors, including gene and predicted amino acidsequences, avian paramyxoviruses deserve their own genus designationamong the Paramyxovirinae.

Newcastle disease (ND) is a contagious viral disease affecting onlyspecies of birds. Clinical signs are extremely variable depending on thestrain of virus, species and age of bird, concurrent disease, andpre-existing immunity.

Vaccination plays a pivotal role in the control of Newcastle disease(ND) in poultry. This can partly be attributed to the fact that severalnaturally derived less pathogenic and attenuated live viral strains wereidentified and have been available for this purpose as early as thesecond half of the 1940's (reviewed by Lancaster, 1964).

The extreme variation in virulence of different ND virus clones and thewidespread use of live vaccines means that the identification of anclone as ND virus from birds showing clinical signs does not confirm adiagnosis of ND, so that an assessment of the virulence of the clone isalso required.

NDV strains have been classified in several ways by several authors andinstitutions. An early form of classification was based on theirpathogenicity, in which the strains were classified into velogenic,mesogenic, lentogenic and avirulent groups (Hanson and Brandly, 1955).

Several potential in-vitro tests for establishing virulence usuallyrelated to the molecular basis for pathogenicity are being investigatedby various groups around the world. At present, a definitive assessmentof virus virulence is usually based on one or more of the followingin-vivo tests, although the current OIE definition allows molecularassessment of virulence.

1. The plaque size and the virulence relationship of the NDV strains waspublished by G. M. Schloer and R. P. Hanson (J Virol. 1968 January;2(1): 40-47.). Schloer and Hanson found that the size of the plaques ofthe NDV was related to virulence for chickens. Markedly larger plaqueswere produced by the velogenic (high virulence) strains while smallerplaques were found in mesogenic (intermediate virulence) strains. Thismethod was used in the past as a way to classify NDV strains bymeasurement of plaque size.

2. Mean death time in eggs The MDT has been used to classify ND virusstrains into velogenic (taking under 60 hours to kill); mesogenic(taking between 60 and 90 hours to kill); and lentogenic (taking morethan 90 hours to kill).

3. Intracerebral pathogenicity index (ICPI). The most virulent viruseswill give indices that approach the maximum score of 2.0, whereaslentogenic strains will give values close to 0.0.The mesogenic strainsfall between 0.7 and 1.5.

4. Intravenous pathogenicity index (IVPI). Lentogenic strains and somemesogenic strains will have IVPI values of 0, whereas the indices forvirulent strains will approach 3.0.

5. Molecular basis for pathogenicity. During replication, ND virusparticles are produced with a precursor glycoprotein, F0, which has tobe cleaved to F1 and F2 for the virus particles to be infectious. Thispost-translation cleavage is mediated by host-cell proteases. Trypsin iscapable of cleaving F0 for all ND virus strains. It would appear thatthe F0 molecules of viruses virulent for chickens can be cleaved by ahost protease or proteases found in a wide range of cells and tissuesthus spreading throughout the host, damaging vital organs, but F0molecules in viruses of low virulence are restricted in theirsensitivity to host proteases resulting in restriction of these virusesto growth only in certain host-cell types. Most ND viruses that arepathogenic for chickens have the sequence ¹¹²R/K-R-Q-K/R-R¹¹⁶ of SEQ IDNO: 4 at the C-terminus of the F2 protein and F (phenylalanine) atresidue 117, the N-terminus of the F1 protein, whereas the viruses oflow virulence have sequences in the same region of ¹¹²G/E-K/R-Q-G/E-R¹¹⁶of SEQ ID NO: 5 and L (leucine) at residue 117. It appears to be therequirement of at least one pair of basic amino acids at residues 116and 115 plus a phenylalanine at residue 117 and a basic amino acid (R)at 113 if the virus is to show virulence for chickens. Based on thesemolecular findings the veterinary classification of ND viruses is nolonger divided into three—but rather into two divisions—pathogenic andapathogenic.

It seems likely that the vast majority of birds are susceptible toinfection with ND viruses of both high and low virulence for chickens,although the clinical signs seen in birds infected with ND virus varywidely and are dependent on factors such as: the virus, host species,age of host, infection with other organisms, environmental stress andimmune status. In some circumstances infection with the extremelyvirulent viruses may result in sudden high mortality with comparativelyfew clinical signs. Thus the clinical signs are variable and influencedby other factors so that none can be regarded as pathognomonic.

Newcastle disease is defined as an infection of birds caused by a virusof avian paramyxovirus serotype 1 (APMV-1) that meets one of thefollowing criteria for virulence:

-   -   A) The virus has an intracerebral pathogenicity index (ICPI) in        day-old chicks (Gallus gallus) of 0.7 or greater    -   B) Multiple basic amino acids have been demonstrated in the        virus (either directly or by deduction) at the C-terminus of the        F2 protein and phenylalanine at residue 117, which is the        N-terminus of the F1 protein. The term ‘multiple basic amino        acids’ refers to at least three arginine or lysine residues        between residues 113 and 116. Failure to demonstrate the        characteristic pattern of amino acid residues as described above        would require characterization of the cloned virus by an ICPI        test. In this definition, amino acid residues are numbered from        the N-terminus of the amino acid sequence deduced from the        nucleotide sequence of the F0 gene, 113-116 corresponds to        residues −4 to −1 from the cleavage site.’

Genetic analyses of NDV strains cloned in the past 80 years haverevealed the existence of at least 9 genotypes (and further subtypes)that showed not only region specific and host species associations buttheir temporal distribution was also apparent (Lomniczi és Czeglédi,2005). It was shown that early genotypes [II.-IV. and Herts'33(W)]prevalent before the 1960s were replaced by recent genetic groups(V.-VIII.) following the introduction of vaccination. Recentlysublineages of the Far East genotype VII have spread to other geographicareas, e.g. to Europe (see family tree of known NDV virus strains (FIG.31)). Replacement of genotypes appears to be an evolutionary processrather than random epidemiological event in the distribution of NDVstrains. The emergence of novel virulent genotypes seems to beinconsistent with the application of vaccination but experimentalinfections shed light on the process whereby immunized chickenpopulation became the reservoir of the novel virulent viruses.

As to the ecology two major reservoirs of NDV strains exist in nature.The primordial reservoir consists of wild water-bird species that harborprimitive (apathogenic) viruses but, surprisingly, only two geneticlineages are known in the wild: class I and genotype I (belonging toclass II). By contrast, the remainder (genotypes II.-VIII.) comprisesvirulent strains and is maintained in the secondary (artificial)reservoirs of chickens. It is hypothesized that the chicken populationswere seeded with apathogenic viruses and pathogenic strains emerged inthe chicken host. Prior to the immunization period at least twoindependent colonizations could have taken place (with genotype I andII).

Genetic analysis of an authentic sample of the first European clone,Herts'33 (cloned in England in 1933), revealed that it represented ahighly diverged novel early lineage. Contrarily to a 1940 publicationfrom England in which the derivation of strain H, one of the mostsuccessful early vaccines, from Herts'33(W) by egg passage was reported,genetic analysis precluded relationships between them.

Genetic analyses of NDV strains have also indicated a remarkable geneticstability of NDV strains, even after prolonged and repeated passage. Thegenetic stability is proven by the lack of viral recombination innature. Toyoda et al. analyzed the sequences of the NH and F genes ofmultiple strains of NDV cloned over a period of 50 years. There was nogene exchange by recombination in the generation of three lineages.(Toyoda T., Newcastle disease virus evolution. II. Lack of generecombination in generating virulent and avirulent strains. Virology169: 273-282, 1989.)

NDV is usually thought to be an avian virus, but it also able to infectshumans. Although NDV causes a potentially fatal, noncancerous disease(Newcastle disease) in birds, it causes only minor illness, manifestedin mild flu like symptoms, or conjunctivitis in exposed humans(historically chiefly observed in laboratory workers).

In 1971 a scientific publication in “The Lancet” by Dr. Laszlo Csatárydescribed case histories of cancer treatment with an undisclosed strainof Newcastle Disease Virus (The Lancet, 1971, 7728, p.825). Subsequentlyto this publication Dr. L. Csatáry and co-workers have published anumber of scientific publications as well as patent applications (seebelow) based on scientific work with a virus strain referred to as“MTH-68/H”. However, none of these references disclose the exact natureof the virus strain, the virus strain has never been commerciallyavailable nor been deposited at any virus library and therefore allthese publications are not enabling for an expert skilled in the art.Moreover, further scientific work exists with virus compositionsreferred to as “MTH-68/H” from scientists other than Dr. L. Csatáry.However, it is highly unclear whether the virus compositions used areidentical to that having been described in the Lancet publication citedabove.

The work of Dr. Csatáry has obviously stimulated the medical communityso that further work has been published such as EP 696326 B1 (WellstatBiologics Corporation) and others as listed below. However, thescientific work disclosed therein cannot be reproduced by an expert inthe art as the strains used therein such as strain 73-T or MK-107 arelikewise not available to the public.

Significant interest has developed in the potential for use of NDV incancer therapy because NDV has been found to have selective cell killingproperties in many types of cancer cells while not effecting normalnon-neoplastic cells. A report indicating that NDV might be useful as acancer treatment was published in the early 1960s. Since then, a numberof studies have been reported.

Many NDV strains have been found to be cytotoxic to cancer cells. Somestrains are able to replicate in and destroy cancer cells while at thesame time not effecting normal cells. These strains have been termedoncolytic. Different strains demonstrate different levels of cancer celldamaging properties, and different cancer cell types show sensitivity todifferent strain types of NDV. These properties define a strain'soncolytic potency. The oncolytic potency is thought to have a clinicalcorrelation as to the therapeutic dosaging requirements. In experimentalconditions the more oncolytically potent the strain is for a cancer celltype the lower the multiples of infective (MOI) viral particles per cellis needed to be able to observe a cytolytic effect. The clinicalimplication is a need for lower viral doses to achieve a therapeuticeffect. While NDV has not been observed to cause any significant diseasein humans—at extremely high therapeutic doses given parentally, as hasbeen used in some clinical trials—it has been noted to potentially causeside effects of hypotension and high fever—leading to the need to findalternative techniques of dosaging—to desensitize the patient prior togiving the high dose application (see WO 00/62735).

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide a new clone of NDVwhich is oncolytic and has improved properties compared to existingstrains.

An expert skilled in the art reading this document will easily identifythe following additional objects of the invention which are summarizedin this section for the reader's convenience. However, the followinglist does not exclude further objects that may be contained in thedescription.

-   -   1) A Newcastle Disease Virus clone which is interferon        insensitive and has an ICPI between 1.2 and 2.0.    -   2) A Newcastle Disease Virus clone according to object 1 which        is interferon insensitive and has an ICPI between 1.2 and 1.5.    -   3) A Newcastle Disease Virus clone according to object 1        comprising the DNA nucleotide sequence of SEQ ID NO: 1.    -   4) A Newcastle Disease Virus clone according to object 1 as        deposited with European Collection of Cell Cultures as Accession        Number 06112101 on Nov. 21, 2006.    -   5) The use of a Newcastle Disease Virus clone according to at        least one of objects 1-4 for the manufacture of a medicament for        the treatment of cancer by causing tumor cell death        -   or for a preventative form of biological cancer therapy,            destroying nascent or residual cancer cells or decreasing            the risk of development of metastatic lesions.    -   6) A use according to object 5, wherein the human tumor cells        are p53-negative human tumor cells.    -   7) A use according to object 5, wherein the human tumor cells        are selected from cervical cancer, ovarian cancer, bladder        cancer, renal carcinoma, Wilm's tumor, prostate cancer, lung        cancer (including bronchial), lymphoma, leukemia, central        nervous system tumors (including meningeoma, medulloblastoma,        glioblastoma, astrocytoma, neuroblastoma); pancreatic cancer,        skin cancer (incl. melanoma), colon cancer, bone (both primary        and metastic lesions) and breast cancer, stomach cancer,        esophageal cancer, thyroid cancer, sarcomas, mesothelioma, head        and neck cancers (including oro-naso-pharyngeal, parathyroid),        hematological malignancies, vulvar, vaginal, endometrial        carcinomas, testicular carcinoma, ano-rectal cancers, hepatic        and extrahepatic (bile duct) cancers, sarcomas (including        Ewings), eye cancer (including retinoblastoma), thymic        carcinoma, urethral cancers, carcinoid tumors or adrenocortical        cancers.    -   8) A use according to object 5, wherein the administration is        provided by way of intravenous, intra-arterial, enteral,        parenteral, intrathecal, intraperitoneal, intrathoracic,        intrapleural, oral, sublingual, bucco-mucosal, intranasal,        intracystic, intraurethral, rectal, vaginal, subcutaneous,        intratumoral, peritumoral, local, intramuscular, intrabronchial,        intrarterial, intracranial, and/or topical application.    -   9) A use according to object 5, wherein a single dose received        by a patient contains 1×10⁵ to 1×10¹² virus particles,        preferably 1×10⁶ to 1×10¹⁰ virus particles, most preferably        1×10⁷ to 1×10⁹ virusparticles.    -   10) A use according to object 5, wherein a patient receives one        or more multiples of 1×10⁵ to 1×10¹² virus particles, preferably        one or more multiples of 1×10⁶ to 1×10¹⁰ virus particles, most        preferably one or more multiples of 1×10⁷ to 1×10⁹ virus        particles frequently between three times a day to once per        month.    -   11) A use according to object 5 in which virus treatment is        combined with chemotherapy, radiotherapy (such as α, β or        γ-radiation, X-ray-radiation, particle-radiation), immunotherapy        or surgery.    -   12) A use according to object 11 in which virus treatment is        applied before, together, or after application of chemotherapy,        radiotherapy (such as α, β or γ-radiation, X-ray-radiation,        particle-radiation), immunotherapy or surgery.    -   13) The use of a Newcastle Disease Virus clone according to at        least one of objects 1 -4 for the manufacture of a medicament        for the treatment of interferon sensitive neoplastic conditions,        especially from Melanoma, (non-Hodgkins) Lymphomas, Leukemias        (Chronic Myeloid Leukemia, Hairy Cell Leukemia), Breast cancer,        Bladder Carcinoma, Renal cell carcinoma, Head and Neck cancer,        Carcinoid tumors, Bile Duct cancers, Pancreatic cancer, Multiple        Myeloma, Kaposi Sarcoma, as well as non-neoplastic interferon        sensitive autoimmune and viral conditions such as Multiple        sclerosis, Condylomata acuminata, Hepatitis, Herpes, Rheumatic        Arthritis, Behcet's Disease, Idiopathic Pulmonary Disease,        Aphthous stomatitis, Severe Malignant Osteoporosis, cervix        cancer, or SARS.    -   14) A method to reduce pain perception in a cancer patient        comprising application of Newcastle Disease virus to said cancer        patient.    -   15) A method to reduce pain perception in a cancer patient        according to object 14 comprising application of Newcastle        Disease virus clone according to at least one of object 1-4 to        said cancer patient.    -   16) A method to reduce side effects of chemotherapy in a cancer        patient treated with chemotherapeutic agents comprising        application of Newcastle Disease virus before, together, or        after the chemotherapeutic agent to said patient.    -   17) A method to reduce side effects of chemotherapy in a cancer        patient treated with chemotherapeutic agents according to object        16 comprising application of a Newcastle Disease virus clone        according to at least one of object 1-4 before, together, or        after the chemotherapeutic agent to said patient.    -   18) A method to reduce side effects of chemotherapy in a cancer        patient treated with chemotherapeutic agents according to object        16 or 17, wherein the side effect is selected from nausea,        vomiting, hair loss, fatigue, loss of appetite intestinal        problems, loss of appetite and weight changes.    -   19) A method to reduce radionecrosis in a cancer patient treated        with radiation comprising the application of a Newcastle Disease        virus before, together, or after radiotherapy to said patient.    -   20) A method to reduce radionecrosis in a cancer patient treated        with radiation according to object 19 comprising the application        of a Newcastle Disease virus clone according to at least one of        object 1-4 before, together, or after radiotherapy to said        patient.    -   21) A method to reduce side effects including acute or chronic        sequelae of radiotherapy in a cancer patient treated with        radiation comprising the application of a Newcastle Disease        virus clone according to at least one of object 1-4 before,        together, or after radiotherapy to said patient.    -   22) A method to reduce side effects of radiotherapy in a cancer        patient treated with radiation according to object 21, wherein        the side effect of radiotherapy is selected from fatigue,        anorexia, skin changes (or other acute symptoms depending on the        exposed organs), diarrhea, incontinence, painful urination,        frequency in urination, difficulty in swallowing, dryness of the        mouth, tenderness, ulceration, cough, shortness of breath, sore        throat, hoarseness, radio-necrosis of the brain tissue or spinal        cord, soft tissue necrosis, osteo-radio-necrosis, subcutaneous        fibrosis, atrophy, telangiestasia, chronic necrosis, unresolved        ulceration, stricture formation, severely decreased saliva        formation, xerostomia, local myelopathies, chondro-necrosis,        ulceration or stricture of the esophagus, fibrosis, pneumonitis        or chronic irritation of the bowel or rectum.    -   23) A method to increase the quality of life in a cancer patient        by amelioration of severe anorexia, loss of energy, depression,        inertia, nausea, or fatigue comprising the application of a        Newcastle Disease virus to said patient.    -   24) A method to increase the quality of life in a cancer patient        according to object 23 comprising the application of a Newcastle        Disease virus clone according to at least one of object 1-4 to        said patient.    -   25) A method of palliative treatment of an advanced cancer        patient comprising the application of a Newcastle Disease virus        to said patient.    -   26) A method of palliative treatment of an advanced cancer        patient comprising the application of a Newcastle Disease virus        clone according to at least one of object 1-4 to said patient.    -   27) A pharmaceutical composition for the treatment of cancer        comprising as an active ingredient a Newcastle Disease virus        clone according to at least one of object 1-4 together with        physiologically acceptable additives.    -   28) A pharmaceutical composition for the treatment of cancer        comprising as an active ingredient a Newcastle Disease virus        clone according to at least one of object 1-4 in freeze dried        form together with physiologically acceptable additives.    -   29) A pharmaceutical kit for the treatment of cancer according        to object 5 comprising a pharmaceutical composition according to        object 27 or 28 and a pharmaceutical composition comprising a        chemotherapeutic agent.    -   30) A pharmaceutical kit for the treatment of cancer according        to object 29 wherein the chemotherapeutic agent is selected        from:        -   alkylating agents, anti-metabolites, anti-tumor antibiotics,            topoisomerase inhibitors, mitotic inhibitors, targeted            therapies, differentiating agents, especially from:        -   arsenic trioxide, adriamycin, BCNU, bexarotene, bleomycin,            carboplatin, cisplatin, decarbazine, doxorubicin,            5-fluorouracil, methotraxate, taxol, temozolomide,            vinblastine, vincristine, azacitidine, azathioprine,            capecitabine, chlorambucil, cyclophosphamide, cytarabine,            daunorubicin, docetaxel, doxifluridine, epirubicin,            epothilone, etoposide, gemcitabine, hydroxyurea, idarubicin,            imatinib, mechlorethamine, mercaptopurine, mitoxantrone,            oxaliplatin, paclitaxel, pemetrexed, sorafenib, teniposide,            tioguanine, tretinoin, valrubicin, vindesine, vinorelbine,            imitanib, gefitinib, erlotinib, sunitinib, bortezomib.    -   31) A method of manufacture of a NDV virus clone according to        objects 1-4, comprising the steps of        -   I. Generating a purified clonal viral clone (e.g. through            multiple plaque purification)        -   II. Inoculating Specific Pathogen Free (SPF) chicken eggs            with the clonal clone        -   III. Incubating the SPF eggs        -   IV. Chilling the SPF eggs        -   V. Harvesting the allantoic fluid from the SPF eggs        -   VI. Removing-debris from the allantoic fluid—possibly using            filtration and or centrifugation        -   VII. Ultracentrifugation of the allantoic fluid.    -   32) A method of manufacture of a NDV virus composition according        to object 31 furthermore comprising the steps of:        -   VIII. Formulating and Filling Individual Containers        -   IX. Lyophilization- and Freeze Drying of Finished Product.    -   33) A Newcastle Disease Virus which is purified and        freeze-dried.    -   34) A Newcastle Disease Virus according to object 33 in which        the Newcastle Disease Virus        -   a) is interferon insensitive and has an ICPI between 1.2 and            2.0, preferably between 1.2 and 1.5        -   or        -   b) comprises the DNA nucleotide sequence of SEQ ID NO: 1        -   or        -   c) is identical to the virus deposited with European            Collection of Cell Cultures as Accession Number 06112101 on            Nov. 21, 2006.

An alternative object of the invention is the use of a Newcastle DiseaseVirus Clone which has an ICPI equal or above 1.4, preferably equal orabove 1.6, most preferably equal or above 1.75 in the treatment ofcancer, the maximum ICPI score being 2.0. Newcastle Disease strainshaving these ICPI values are known in the prior art and are availablefrom different sources such as the Central Veterinary Laboratory, NewHaw Addlestone, United Kingdom.

Strains of the above identified property (ICPI score) include thestrains Mukteswar, Roakin, Beaudette C, G B Texas, N Y Parrot 701811972, Italien, Milano, Herts '33/56. These strains can be obtained fromsources in Yugoslavia, India, USA, Portugal and Italy.

As mentioned above these strains are known and obtainable from publicsources. They have been used for experimental purposes (especially inanimal diseases) and for vaccination procedures in poultry). However,these strains have never been used for the manufacture of drugs for thetreatment of human diseases, such as cancer. A preferred group ofstrains is interferon-insensitive. “Interferon insensitivity” issupposed to mean that cell proliferation does not change in the presenceof interferon compared to the absence of interferon.

It has turned out, that the use of Newcastle Disease Virus (NDV) strainshaving an ICPI of 1.4 and above are very effective in treatment of humancancer. Newcastle Disease Virus strains having an ICPI of 1.4 These NDVstrains are effective in the treatment of cancer by causing tumor celldeath. They are moreover effective for a preventative form of biologicalcancer therapy, destroying nascent or residual cancer cells ordecreasing the risk of development of metastatic lesions.

The virus strains can be used to treat the various kinds of cancermentioned earlier in this application (see object No. 13). They can beused as the sole treatment or in combination with chemotherapy, radiotherapy, immunotherapy or surgery. They can be administered as describedearlier in this application (see object No. 7). A single dose receivedby a patient generally contains 1×105 to 1×1012 virus particles,preferable 1×106 to 1×1010 virus particles, most preferably 1×107 to1×109 virus particles. A patient may receive one or more doses (frequentadministration between three times a day to once per month), (see objectNos. 9 and 10).

Treatment of patients with the virus strains disclosed above results inimprovement of their respective cancer disease. In addition saidtreatment usually reduces the pain perception in cancer patients. Itfurthermore reduces side effects of chemotherapeutic agents (whenapplied in combination with such chemotherapeutic agent, such as nausea,vomiting, hair loss, fatigue, loss of appetite, intestinal problems, andweight changes), (see object Nos. 14-26)

Further details of application forms, manufacturing processes,pharmaceutical compositions may be found earlier in this document. Themethods described there may be easily adapted to the needs of NewcastleDisease strains having an ICPI of 1.4 and above.

It has been found that a new clone of NDV which is interferoninsensitive and which has an ICPI between 1.2 and 2.0 is particularlybeneficial in cancer therapy having properties which have not beenobvious to experts skilled in the art.

A particularly beneficial NDV strain for cancer therapy is referred toas “MTH-68H/VB”, as studies have indicated that this NDV clone may besuccessful in destroying tumors when other cancer therapies, includingother NDV strains and their formulations have failed. This beneficialnew NDV strain is variously referred to herein as MTH-68H/VB,MTH-68/H-VB, MTH-68/HVB, MTH-68/VB, MTH-68/H/VB and MTH-68/HVB, all ofwhich designations refer to the very same NDV strain. Its inherent highpotency allows MTH-68/H-VB to possibly be given at lower effectivedoses; making it also possible to be applied using less invasive nonintravenous forms of administration. MTH-68/H-VB has been demonstratedto have a markedly synergistic effect with other modes of cancertherapy, especially radiation therapy—while seeming to have acyto-protective effect on normal cells, thus making it an idealcandidate not only as a sole form of cancer therapy, but also as an oncotherapy to be used as an adjunct to other more traditional therapies.

Surprisingly, it has been found that a Newcastle Disease Virus clonewhich is interferon insensitive and has an ICPI between 1.2 and 2.0(preferably between 1.2 and 1.5) shows superior oncolytic propertiescompared to previous strains.

More particularly, it has been found that a Newcastle Disease Virusclone which is comprising the DNA nucleotide sequence of SEQ ID NO: 1 isan example of such a Newcastle Disease Virus clone. Also, it has beenfound that the virus deposited with European Collection of Cell Culturesas Accession Number 06112101 is an example of such a Newcastle DiseaseVirus clone.

The term clone as used herein refers to viruses that are geneticallyhomogeneous as it has been found that previous virus compositions usedin the art are genetically heterogenous.

Surprisingly, a particular new clone of NDV named as MTH-68/H-VB, hasbeen found to have superior oncolytic properties. The new NDV clone ischaracterized

-   -   (a) By its nucleotide and predicted amino acid sequences in the        fusion (F) protein gene. Protein sequence at the C-terminus of        the F2 protein and, the N-terminus of the F1 protein. Amino acid        residues are numbered from the N-terminus of the amino acid        sequence deduced from the nucleotide sequence of the F0 gene,        113-116 corresponds to residues −4 to −1 from the cleavage site    -   (b) By its plaque size, shape and appearance    -   (c) By the ICPI of the clone    -   (d) By its uniquely significant interferon induction capacity    -   (e) By its unique interferon insensitivity and    -   (f) By its ability to destroy tumour cells under different        conditions    -   (g) By its compelling cancer associated pain-reducing properties    -   (h) By its potent synergistic potentiating effect on other modes        of cancer treatments; including chemotherapy and radiation        therapy.    -   (i) By its ability to lessen the deleterious side effects of        chemotherapy and radiation therapy—thereby rendering more        applicable these other traditional onco-therapeutic modalities.    -   (j) By the full nucleotide sequence which is disclosed in SEQ ID        NO: 1 (Annex).    -   (k) By the cloned culture deposited on November 21^(st), 2006        with the European Collection of Cell Cultures as Accession        Number 06112101.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the two sequences of the F gene, namely, thenucleotide sequence of SEQ ID NO: 2 and the amino acid sequence of SEQID NO: 3.

FIG. 2 illustrates the plaques of the MTH-68 H/VB clone (resulting fromthe test according to Schloer/Hanson.

FIG. 3 illustrates the plaques before plaque purification.

FIG. 4 is a comparison of virulence and cytopathology of various NDVstrains.

FIG. 5 illustrates the interferon induction of different NDV strains.

FIG. 6 illustrates the IFN-beta effect on the growth of HEK293 cells(Exp. A).

FIG. 7 illustrates the IFN-beta effect on the growth of HEK293 cells(Exp. B).

FIG. 8 illustrates the effect of interferon-beta on the cytotoxicity ofMTH-68H/VB (MTH) in HEK293 cells (Exp. A).

FIG. 9 illustrates the effect of interferon-beta on the cytotoxicity ofMTH-68H/VB (MTH) in HEK293 cells (Exp. B).

FIG. 10 illustrates the effect of anti-IFN-beta on the growth of F11primary human fibroblast cells (Exp. C).

FIG. 11 illustrates the effect of anti-IFN-beta on the cytotoxicity ofMTH-68H/VB in F11 primary human fibroblasts (Exp. D).

FIG. 12 illustrates the effect of anti-IFN-beta on the cytotoxicity ofMTH-68H/VB in F11 primary human fibroblasts (Exp. C).

FIG. 13 illustrates the effect of anti-IFN-beta on the cytotoxicity ofMTH-68H/VB in F11 primary human fibroblasts (Exp. D).

FIG. 14 illustrates the cytotoxic effect of MTH-68H/VB on different celllines.

FIG. 15 illustrates the cytotoxic effect of MTH-68H/VB on different celllines.

FIG. 16 illustrates the cytotoxic effect of different NDV strains(H-fibroblast cell line).

FIG. 17 illustrates the cytotoxic effect of different NDV strains (HeLacell line).

FIG. 18 illustrates the cytotoxic effect of different NDV strains (293cell line).

FIG. 19 illustrates the cytotoxic effect of MTH-68H/VB on BC-1, HT-3 and293 cells.

FIG. 20 illustrates the effect of MTH-68H/VB on DAOY, SK-N-FI and IMR-32cells.

FIG. 21 illustrates the effect of MTH-68H/VB on OVCAR cells.

FIG. 22 illustrates the effect of MTH-68H/VB on C-41 cells.

FIG. 23 illustrates the effect of MTH-68H/VB treatment on the growth ofsubcutaneous GI261 tumors (weekly treatment).

FIG. 24 illustrates the effect of MTH-68H/VB treatment on the growth ofsubcutaneous GI261 tumors (daily treatment).

FIG. 25 illustrates the combined effect of MTH-68H/VB treatment andtumor irradiation on the growth of subcutaneous GI261 tumors.

FIG. 26 illustrates the survival of tumor bearing mice after combinedtreatment with MTH-68H/VB and irradiation.

FIG. 27 illustrates tumor growth rate after combined treatment withirradiation, temozolomide and MTH-68H/VB—experiment 1.

FIG. 28 illustrates tumor growth rate after combined treatment withirradiation, temozolomide and MTH-68H/VB—experiment 2.

FIG. 29 illustrates the effect of MTH-68H/VB, irradiation and BCNUtreatment on the tumor size in tumor bearing mice.

FIG. 30 illustrates the survival of tumor bearing mice (%).

FIG. 31 illustrates the family tree of known NDV virus strains.

FIGS. 32 and 33 show details of the genes and open reading frames forMTH-68H/VB.

DETAILED DESCRIPTION OF THE INVENTION

One NDV clone according to the invention may be described as follows:

1. DESCRIPTION OF THE NEW CLONE BY THE CHARACTERIZATION OF THE GENOME OFMTH-68H/VB VIRUS STRAIN BY SEQUENCING

The genome was amplified in five overlapping portions. Three sequencespecific primers were used for the RT and three primer pairs for theamplification of the inner regions encompassing 94% of the genomeamplified in five overlapping portions.

The genomic RNA sequence of MTH-68/VB consists of 15186 nt. The 3’ and5′ ends of the genome comprise the leader and trailer regions. Theleader sequence is 55 nt long while the trailer sequence is 113 nt inlength in MTH-68/VB. NDV strains have 114 nt trailer region in general.

The negative-strand RNA genome of MTH-68/VB contains six genes encodingsix major structural proteins in 3′→5′ direction (3′NP-P-M-F-HN-L 5′)and two additional non-structural proteins (V and W). Structuralproteins are NP: 489 aa (amino acids), P: 395 aa, M: 364 aa, F: 553 aa,HN: 571 aa, L: 2204 aa in length respectively.

During transcription one or two non-templated G residues can be insertedat the conserved transcriptional editing-site (UUUUUCCC) of SEQ ID NO: 6within the P gene, producing two alternative ORFs. The phosphoprotein,the V(+1 frame) and the W(+2 frame) share the amino-terminal and vary attheir carboxy-terminal ends in length and amino acid composition[Steward et al. 1993]. The sequence of the transcriptional editing siteof MTH-68/VB is 2281UUUUUCCC2288 of SEQ ID NO: 7. The ORF of the Vprotein is 717 nt in length from nt position 1888 to 2607 of the genomicRNA sequence and encodes a protein of 239 aa residues in strainMTH-68/VB. The ORF of the W protein is 681 nt long from nt position 1888to 2571 of the genomic RNA sequence and encodes a protein of 227 aa inlength.

Length of intergenic regions of MTH-68/VB ranges from 1 to 47. The NP-P,P-M, and M-F junctions are only one nt, intergenic sequence between F-HNgenes consist of 31 nt, whereas HN-L junction is 47 nt long.

Details of the genes and open reading frames are summarized in FIGS. 32and 33 and map of the complete genomic RNA of MTH-68/VB is described inthe attached sequence listing (SEQ ID NO: 1) as hard-copy. With respectto FIG. 32, the following sequences and their corresponding numbers inthe attached sequence listing are illustrated: UGCCCAUCUU of SEQ ID NO:8; AAUCU₇ of SEQ ID NO: 9; UGCCUGUGUU of SEQ ID NO: 10; AAUUCU₆ of SEQID NO: 11; UGCCCAUCUU of SEQ ID NO: 12; AAUCU₆ of SEQ ID NO: 13;UGCCCAUCUU of SEQ ID NO: 14; AAUUCU₆ of SEQ ID NO: 15; GAUGGCCUACAUCCUCUUGU UUUCCCUUAU A of SEQ ID NO: 16; UGCCCAUCUU of SEQ ID NO: 17;AAUUCU₆ of SEQ ID NO: 18; ACACCUGCCA CCACUUUAUG UUCCGUUUUG UCGAGUGUCUGGUGUCG of SEQ ID NO: 19; UGCCCAUCCU of SEQ ID NO: 20; AAUCU₇ of SEQ IDNO. 21. With respect to FIG. 33, the following sequences and theircorresponding numbers in the attached sequence listing are illustrated:UGGUUUGUCU CUUAGGCACU CUAUGCUAUU UUCCGCUUCC UCGUUAGCUU CAGCU of SEQ IDNO: 22; UGCCCAUCUU of SEQ ID NO: 23; AAUCUUUUUUU of SEQ ID NO: 24;UGCCUGUGUU of SEQ ID NO: 25; AAUUCUUUUUU of SEQ ID NO: 26; UGCCCAUCUU ofSEQ ID NO: 27; AAUCUUUUUU of SEQ ID NO: 28; UGCCCAUCUU of SEQ ID NO: 29;AUCUUUUUU of SEQ ID NO: 30; GAUGGCCUAC AUCCACUUGU UUUCCCUUAU A of SEQ IDNO: 31; UGCCCAUCCU of SEQ ID NO: 32; CAAUCUUUUUU of SEQ ID NO: 33;ACACCUGCCA CCACUUUAUG UUCCGUUUUG UCGAGUGUCU GGUGUCG of SEQ ID NO: 34;UGCCCAUCCU of SEQ ID NO: 35; AAUCUUUUUUU of SEQ ID NO: 36; UAACUUAAGGCUGGGAAAUU CUGAGCAUAA GCCUAAGUUU AUUAAUAGAG UCUUUUUUUC ACGUGCAUCAACAAGAACUA AUAUCAGGGC AGUAAGUGGU UUAGAAACAA ACCA of SEQ ID NO: 37.

One object of the invention is therefore a Newcastle Disease Virus clonecomprising the DNA nucleotide sequence of SEQ ID NO: 1.

2. DESCRIPTION OF THE VIRULENCE AND PATHOGENICITY OF THE NEW CLONE BYTHE DETERMINATION OF ITS NUCLEOTIDE AND PREDICTED AMINO ACID SEQUENCESIN THE FUSION (F) PROTEIN GENE

The new clone MTH-68H/VB has been characterized by its nucleotide andpredicted amino acid sequences in the fusion (F) protein gene, Proteinsequence at the C-terminus of the F2 protein and, the N-terminus of theF1 protein. Amino acid residues are numbered from the N-terminus of theamino acid sequence deduced from the nucleotide sequence of the F0 gene,113-116 corresponds to residues -4 to -1 from the cleavage site

During replication, ND virus particles are produced with a precursorglycoprotein, F0, which has to be cleaved to F1 and F2 for the virusparticles to be infectious. This post-translation cleavage is mediatedby host-cell proteases. Trypsin is capable of cleaving F0 for all NDvirus strains.

It would appear that the F0 molecules of viruses virulent (for chickens)can be cleaved by a host protease or proteases found in a wide range ofcells and tissues, and thus spread throughout the host damaging vitalorgans, but F0 molecules in viruses of low virulence are restricted intheir cleavability to certain host proteases resulting in restriction ofthese viruses to growth only in certain host-cell types.

Most ND viruses that are virulent (pathogenic for chickens) have thesequence ¹¹²R/K-R-Q-K/R-R¹¹⁶ of SEQ ID NO: 4 at the C-terminus of the F2protein and F (phenylalanine) at residue 117, the N-terminus of the F1protein, whereas the viruses of low virulence have sequences in the sameregion of ¹¹²G/E-K/R-Q-G/E-R¹¹⁶ of SEQ ID NO: 5 and L (leucine) atresidue 117. Thus there appears to be the requirement of at least onepair of basic amino acids at residues 116 and 115 plus a phenylalanineat residue 117 and a basic amino acid (R) at 113 if the virus isregarded velogenic (showing virulence for chickens; OIE Manual)]

Based on the partial sequence of the fusion gene (F) between nucleotidepositions 47 and 420, the MTH-68/VB strain has virulent sequence ofproteolytic cleavage site due to the dibasic motif and an F aa atposition 117.(see in red). Cleavage occurs between 116 and 117 aminoacids.

It is therefore an object of the invention to provide a NewcastleDisease Virus clone having a virulent sequence of proteolytic cleavagesite with a dibasic motif and an F aa at position 117. More particularlyit is an object of the invention to provide a Newcastle Disease Virushaving a sequence with at least one pair of basic amino acids atresidues 116 and 115 plus a phenylalanine at residue 117 and a basicamino acid at 113.

3. DESCRIPTION OF THE VIRULENCE AND PATHOGENICITY OF THE NEW CLONE BYTHE DETERMINATION OF ITS PLAQUE FORM AND SIZE BY THE TEST DESCRIBED BYSCHLOER AND HANSON (JOURNAL OF VIROLOGY, 2 (1968), P. 40-47).

The authors of this publication did believe in 1968 that the ability ofthe Newcastle disease virus strains to produce large plaques is relatedto their virulence in chickens. They compared the plaque-size understandard conditions in chicken embryo fibroblast cell monolayers.Markedly larger plaques were produced by the velogenic (high virulence)strains. Only small plaques were created by the mesogenic (intermediatevirulence) strains, and plaques were rarely found in lentogenic (lowvirulence) strains.

Although this test has never been accepted in the scientific communityas a criteria in the description of NDV virus strains; beside the otherparameters measured, we also studied the plaque forming ability of ournew NDV clone.

The monolayer cultures of chicken embryo fibroblast (CEF) used for theplaque assay were prepared from 10-day-old SPF chicken embryos. Cellmonolayers were grown in petri dishes (Anumbra) 5 cm in diameter(3.5×10⁶ cells per dish) and were infected by the clone virus propagatedin SPF chicken embryos. Plaque formation in CEF culture by theMTH-68H/VB strain is shown in FIG. 2. The plaques formed within 48 to 72h by the clone strains were regarded as “large plaques”. Plaques of thestrain were well-defined, circular. The plaques first appeared at 36 to48 hr after infection and measured between 2.5 to 4.9 mm on the fifthday after inoculation. Microscopically their borders were not clearlydefined. According to the results of the specific test first describedby Schloer and Hanson the new clone can be regarded as velogenic NDV andwould not be considered a mesogenic strain

4. DESCRIPTION OF THE ORIGIN OF THE NEW CLONE AND THE DIFFERENCE BETWEENTHE NEW CLONE AND THE PARENT VIRUS STOCK

The parent strain of the MTH-68H/VB was the “ancient” NDV poultryvaccine strain originated from UK (in the 1940-ies) which had remarkablegenetic heterogeneity. To increase the homogeneity of the product andremove any defective particles plaque purification steps were performedmultiple times. Plaque purification is a technique commonly used inthose skilled in the art to obtain a clonal virus clone that holdsdesired characteristics typified by plaque size-shape and appearance(e.g. Massaab et al, In Plotkin and Mortimer, eds Vaccines,Philadelphia: WB Saunders Co., 1994 pages 78-801.The first partialpurification resulted in a visually homogeneous virus population. Duringthe propagation in CEF the plaques formed by the “purified” virus stockstill showed a great variability (FIG. 3).From this virus populationpropagated in chicken fibroblast monolayer tissue culture one plaquecontaining desired characteristics from the several still variousplaques was chosen and separated and further propagated. At a laterdate, this homogeneous virus population was again propagated in CEF andagain underwent plaque purification where a single plaque was furtherchosen (third isolation) based on its size and appearance and propagatedin SPF chicken embryos: After strict quality control the harvested virussuspension was used for the production of a new master seed ofMTH-68H/VB

5. DESCRIPTION OF THE VIRULENCE AND PATHOGENICITY OF THE NEW CLONE BYTHE DETERMINATION OF ITS INTRACEREBRAL PATHOGENICITY INDEX (ICPI)

Newcastle Disease Viruses are divided into three different pathotypescharacterized by the intracerebral pathogenicity index (ICPI).

Lentogenic (low virulent) strains have an ICPI of 0-0.7. Mesogenicstrains are NDVs of moderate pathogenicity (ICPI 0.7-1.5) and velogenic(high pathogenic) strains are characterized by an ICPI>1.5. The maximumICPI is 2.0.

The ICPI is determined by intracerebral injection of 50 μl of a virusdilution (haemagglutination titre (HA) of at least 24) in day-oldchicks. The animals are observed for 8 days and are assessed once daily(healthy=0, ill=1, dead=2). The ICPI is calculated by dividing the sumby the number of assessments.

It has been found that a Newcastle Disease Virus having a ICPI betweenabout 1.2 and 2.0, preferably between 1.2 and 1.5 does have improvedoncolytic properties, especially when it is interferon insensitive (seebelow).

With the classical technique the ICPI of MTH-68/H-VB was found to fallbetween 1.2 and 1.5, tending towards increased virulence.

6. DESCRIPTION OF THE UNIQUE INTERFERON INDUCTION CAPACITY OF THE CLONE

Interferons (IFNs) are natural cell-signaling proteins produced by thecells of the immune system of most vertebrates in response to challengessuch as viruses, parasites and tumor cells. Interferons are produced bya wide variety of cells in response to the presence of double-strandedRNA, a key indicator of viral infection.

Interferon production has been stimulated by a variety of RNA- andDNA-containing viruses, including those with oncogenic properties suchas polyoma and Rous sarcoma virus.

To investigate the interferon induction of different NDV strains (listedin FIG. 4) there were three system used.

The virus content of the chicken embryo (allantois cavity) or in chickenembryo fibroblast (CEF) cultures was inactivated by heat (65 C° for 30minutes) and the cultures were tested for the presence of interferon byan assay based on the reduction of cytopathic effect of a challengevirus (Sindbis).

In human and swine cell line the interferon induction was measured bythe same technique (described by Falcoff at al. 1966) but for by theassay based on the reduction of cytopathic effect of a challenge virus(Sindbis) the amnion (human) and PK-15 (swine) cell line were used.

On the basis of our data we could conclude that all the studiednon-inactivated virus strains could induce interferon production in alarge magnitude. The most pronounced interferon production was seen inthe PK-15 (pig kidney) and in the human (amnion membrane) cell line(FIG. 5). These results suggest that interferon induction by NDVinvolves components of the virion.

The interferon induction of the new clone MTH-68H/VB was significantlyhigher and more consequent in all the systems tested, which includedseveral different NDV strains of varying virulence, using severaldifferent cell lines—under several different conditions.

It is an object of the invention to provide a NDV virus clone whichsignificantly induces interferon in cells. Due to this behavior the NDVclone may successfully be used to treat diseases which have been shownto be interferon sensitive, such as Melanoma, (non-Hodgkins) Lymphomas,Leukemias (Chronic Myeloid Leukemia, Hairy Cell Leukemia), Breastcancer, Bladder Carcinoma, Renal cell carcinoma, Head and Neck cancer,Carcinoid tumors, Bile Duct, Pancreatic ca, Multiple Myeloma, KaposiSarcoma, as well as non-neoplastic interferon sensitive autoimmune andviral conditions such as Multiple sclerosis, Condylomata acuminata,Hepatitis, Herpes, Rheumatic Arthritis, Behcet's Disease, IdiopathicPulmonary Disease, Aphthous stomatitis, Severe Malignant Osteoporosis,cervix cancer, or SARS (Severe Acute Respiratory Syndrome).

Because of the oncolytic and significant interferon inducing propertiesof the NDV clones according to the invention (especially MTH-68/HVB) itcould be an ideal candidate to be used as part of cancer vaccine typetherapies where it is combined with a patient's autologous tumor cells,and used in combination with other immune modulating factors as part atargeted immuno onco-therapy.

7. DESCRIPTION OF THE UNIQUE INTERFERON NON-SENSITIVITY OF THE CLONE

It is known that most of the viruses including the majority of the NDVstrains are sensitive to interferon β (IFNβ). IFBβ inhibits thereplication of the interferon sensitive NDV in sensitive cells. One canexpect the cytotoxic effect of the interferon sensitive NDV strains todecrease with increasing IFNβ concentration.

We investigated whether the MTH-68H/VB clone is sensitive to interferonβ and the presence of interferon β can modify the virus replication invarious cell lines that are sensitive to MTH-68H/VB infection.

In the first set of experiments MTH-68H/VB sensitive HEK293 cell lineswere infected at various multiplicity of infection (MOI, virus/cells)with MTH-68H/VB in the presence of different concentrations of IFNβ.

In the second set of experiments MTH-68H/VB resistant primary humanfibroblast cells were treated with different concentrations of the virusin the presence of anti-IFNβ. Under these conditions the cytotoxiceffect of MTH-68H/VB should have increased, if IFNβ influenced the viralreplication.

During the studies the HEK293 cell line (human embryonic kidneytransformed with adenovirus type 5) and F11 (primary human fibroblast)cell line were used. Both cell lines were cultured in Dulbecco'sModified Eagle Medium containing antibiotics (penicillin, streptomycinand amphotericin B) and fetal calf serum (10% and 20% for HEK293 and F11cells, respectively). 2×10³ cells were plated on 96 well culture dishesin 100 μl final volume. Twenty-four hours later the cells were infectedwith MTH-68H/VB at different multiplicity of infection (MOI). Cells werealso treated either with IFNβ (R and D Systems) or with an antibodyagainst IFNβ (R and D Systems). Seventy-two hours after NDV infection 20μl WST-1 reagent (Roche) was added and cytotoxicity was assayed 2 hrlater by measuring OD₄₉₀ values with a multi-well photometer.

-   -   Exp. A. HEK293 cells were plated on day 0. Cells were treated        with 20, 200 or 2000 U/ml IFNβ on day 0, 1, 2 and 3. MTH-68H/VB        infection was performed on day 1 at 0.001 or 0.01 MOI.        Cytotoxicity was measured on day 4.    -   Exp. B. Experimental conditions were similar to Exp A, except        that the cells were infected at 0.01 and 0.1 MOI.    -   Exp. C. F11 cells were plated on day 0. Cells were treated with        2, 10, 20 μg/ml anti-IFNβ on day 0, 1, 2 and 3. MTH-68H/VB        infection was performed on day 1 at 0.001 or 0.01 MOI.        Cytotoxicity was measured on day 4.    -   Exp. D. Experimental conditions were similar to Exp C, except        that the cells were infected at 1 or 10 MOI. The final        concentration of anti-IFNβ was 2 and 15 μg/ml.

Effect of IFNβ treatment on the cytotoxic effect of MTH-68H/VB: When theresults were analyzed it was concluded that

-   -   IFNβ did not affect cell proliferation (FIGS. 6 and 7).

IFNβ does not inhibit the cytotoxic effect of MTH-68H/VB

-   -   -   The effect of IFNβ on the cytotoxicity of MTH-68H/VB was            investigated on HEK293 cells using two replication of the            study.(Exp. A and B). In both study the MTH-68H/VB was            cytotoxic for HEK 293 cells at low MOI (lower than 1 MOI)            showing that MTH-68H/VB can replicate in this cell line            (FIGS. 8 and 9, columns 1-3), and as a final conclusion we            proved that IFNβ does not inhibits the cytotoxic effect of            MTH-68H/VB, not even at very high IFNβ concentrations (FIG.            9, columns 4-12)

It is therefore an object of the invention to provide a NDV virus clonethat is insensitive to interferons, especially Interferon R. Interferoninsensitivity is supposed to mean that cell proliferation does notchange in the presence of interferon compared to the absence ofinterferon.

The Effect of IFNβ Antibody Treatment on the Cytotoxicity of MTH-68H/VBin Primary Human Fibroblast Cells

Next, we analyzed the effect of an antibody raised against IFNβ on thecytotoxic effect of MTH-68H/VB in F11 primary human fibroblast cells.Anti-IFNβ did not affect the proliferation of F11 cells in twoindependent experiments (Experiments C and D, FIGS. 10 and 11). Usingtwo different stocks of MTH-68H/VB we could not detect a cytotoxiceffect on fibroblast cells (FIGS. 12 and 13, columns 1-3). Neither low,nor high concentrations of anti-IFNβ treatments were able to increasethe cytotoxic effect of MTH-68H/VB (FIG. 12, columns 4-12; and FIG. 13,columns 4-9).

There were indications that the cytotoxic effect of Newcastle diseasevirus (NDV) relies on the interferon production of the cell lines.According to this suggestion cells (usually normal cells) capable toshow a strong interferon response are resistant to NDV infection, whiletumor cells which lost their IFN response are sensitive to the cytotoxiceffect of NDV. If this hypothesis is correct then the addition of IFNβto tumor cells will restore their resistance against NDV. On the otherhand by removing IFNβ from the neighborhood or culture media of normalcells (fibroblast, for instance) will increase their sensitivity to NDVinfection. Our data contradicts this hypothesis, at least in or case ofMTH-68H/VB, in the experimental models described.

IFNβ treatment of MTH-68H/VB sensitive HEK293 cells did not improvetheir resistance against MTH-68H/VB infection. Besides this, the removalof IFNβ from the culture medium of primary human fibroblast cells byantibody treatment did not increase their sensitivity to MTH-68H/VB. Ourdata demonstrates that the cytotoxic effect of MTH-68H/VB did not dependon the interferon production of the infected cells.

8. COMPARATIVE STUDIES OF THE CYTOTOXIC EFFECT OF MTH-68/VB AND OTHERNDV STRAINS ON VARIOUS IN VITRO GROWING CELL LINES (INCLUDING HUMAN ANDRODENT BRAIN TUMOR CELL LINES, AS WELL AS NORMAL PRIMARY HUMANFIBROBLAST CELLS)

8.1. Replication of MTH-68/H-VB in Different Cell Lines

8.1.1. The Cytotoxic Effect of MTH-68H/VB was Studied Under In VitroConditions in Different Primary and Established Cell Lines Using 20%Fetal Calf Serum.

In this study the following cell lines were used:

-   -   293N3S—human embryonic kidney transformed with adenovirus type        5, adapted to grow in suspension culture (bought from ATCC)    -   HeLa—human epithelial cell line established from a cervix tumor        (bought from ATCC)    -   9L—established rat glioma cell line (bought from ECACC)    -   H-primary human fibroblast cell line (established from skin        biopsy)    -   V-primary human fibroblast cell line (established from skin        biopsy)    -   A-primary human fibroblast cell line (established from skin        biopsy)

The cells were grown in the presence of 20% fetal calf serum, that isthe optimal serum concentration for the primary human fibroblast cells.At this high serum concentration MTH-68H/VB had no cytotoxic effect onnormal human fibroblasts. It had only moderate effect on rat (9 L)glioma cells (FIG. 8. about 50% survival at 50 MOI). Contrary to this,293N3S and HeLa cells were very sensitive to MTH-68H/VB treatment. Even,after infection at a very low MOI (0.005), all cells were killed by thevirus.

The cytotoxicity assays suggested that MTH-68/H-VB could replicate in293N3S and HeLa cells, but no infectious viral particles were formed inthe other investigated cell lines.

8.1.2. The Cytotoxic Effect of MTH-68H/VB on Different Cell Lines UnderIn Vitro Conditions Using 10% Fetal Calf Serum.

To further study potential virus replications, the following cell lineswere used:

-   -   293—human embryonic kidney transformed with adenovirus type 5        (bought from ATCC)    -   HeLa—human epithelial cell line established from a cervix tumor        (bought from ATCC)    -   H-primary human fibroblast cell line (established in our lab        from skin biopsy)    -   961107 primary human fibromatous meningeoma cells established at        NINS    -   960612 primary human fibromatous meningeoma cells established at        NINS    -   980128/2 primary human glioblastoma multiforme cells established        at NINS

5×10³ cells were plated on 96 well culture dishes in 100 μl finalvolume. Twenty-four hours later the cells were infected with thecorresponding virus at different multiplicity of infection (MOI,virus/cell ratio: 0; 0.01; 0.1; 1; 10; 100) by adding 50 μl completemedium containing the virus. Seventy-two hours later 15 μl WST-1 (Roche)was added and cytotoxicity was assayed 1 hr later by measuring OD450values with a multi-well photometer. The OD₄₅₀ values were converted tosurvival rates (%).

It is well known, that high serum concentrations might inhibit theinfectivity of certain viruses (for instance adenovirus). Therefore, inthese experiments only 10% fetal calf serum was added to the cellculture medium. This serum concentration is optimal for all cell lines,except for the primary fibroblasts, but primary human fibroblast cellsare also able to grow in this medium. In the presence of 10% serumMTH-68H/VB at high MOI (10 and 100) was toxic for all cells but slightlyfor primary human fibroblast cells (H-fibroblasts, FIG. 15). The variousprimary human brain tumor cell lines (980128/2, 960612 and 961107)showed much higher sensitivity to MTH-68H/VB than the primaryfibroblasts.

Beside the brain tumor and fibroblast cell lines, we also tested thesensitivity of HeLa cells to MTH-68H/VB treatments. HeLa cells werealmost as sensitive as 293 cells, suggesting that the virus canreplicate in these cells, as well.

8.2. Cytotoxicity of MTH-68/H-VB and Other NDV Strains in different CellLines

In this comparative study the cytotoxicity of different NDV strains werecompared to the cytotoxicity of MTH-68H/VB. In the study the NDV strainswere:

-   -   H/W—the Weybridge line of strain Hertfordshire    -   Mukteswar (“mesogenic” strain from Veterinarski Zavod, Subotica)    -   LaSota—the standard poultry vaccine strain    -   VP—the “avirulent” standard vaccine strain

The comparative cytotoxicity was studied on the 293, HeLa andH-fibroblast cell lines.

-   -   293—human embryonic kidney transformed with adenovirus type 5        (bought from ATCC)    -   HeLa—human epithelial cell line established from a cervix tumor        (bought from ATCC)    -   H-primary human fibroblast cell line (established in our lab        from skin biopsy)

For the assay 5×10³ cells were plated on 96 well culture dishes in 100μl final volume. Twenty-four hours later the cells were infected withthe corresponding virus at different multiplicity of infection (MOI,virus/cell ratio: 0; 0.01; 0.1; 1; 10; 100) by adding 50 μl completemedium containing the virus. Seventy-two hours later 15 μl WST-1 (Roche)was added and cytotoxicity was assayed 1 hr later by measuring OD450values with a multi-well photometer. The OD450 values were converted tosurvival rates (%)

8.2.1. Comparative Study on H-Primary Human Fibroblast Cell Line.

In these experiments 10% fetal calf serum was added to the cell culturemedium. This serum concentration is optimal for all cell lines, exceptfor the primary fibroblasts, but primary human fibroblast cells are alsoable to grow in this medium. In the presence of 10% serum MTH-68H/VB wasslightly toxic for the fibroblast cell but only at very high MOI (10 and100, FIG. 16).

The most pronounced effect was seen in the MTH-68H/VB treated cells,less but still perceptible effect was caused by the H/W strains whilethe others had no cytotoxic effect.

It can be concluded that under normal circumstances, ie unless appliedat very high multiples of infection, the studied NDV strains are notcytotoxic to normal human cells.

8.2.2. Comparative Study on HeLa (Human Epithelial Cell Line Establishedfrom a Cervix Tumor) Cell Line

In these experiments also 10% fetal calf serum was added to the cellculture medium. This serum concentration is optimal for all cell lines.In the presence of 10% serum MTH-68H/VB was highly toxic for the HeLacells even in very low MOI (FIG. 11). The cytotoxic effect of MTH-68H/VBwas significant higher than any other studied viruses especially at lowMOI (0.01 and 0.1).

The sensitivity of HeLa cells suggest that only some of the ND virusstrains can replicate in these cells. Among the studied strainsMTH-68H/VB is the most effective.

8.2.3. Comparative Study on 293 (Human Embryonic Kidney Transformed withAdenovirus Type 5) Cell Line

Similarly to the previous experiment, in the presence of 10% serumMTH-68H/VB was highly toxic for the 293 cells, even at very low MOI(FIG. 12). The cytotoxic effect of MTH-68H/VB was significant higherthan any other studied viruses especially at low MOI (0.01 an 0.1).

The sensitivity of 293 cells suggests that only some of the ND virusstrains can replicate in these cells. Among the studied strainsMTH-68H/VB is the most effective.

8.3. The Cytotoxic Effect and the Potential Replication of MTH-68H/VBStrain in Different Cell Lines of Human Tumor Origin

Cell Lines

Brain Tumors:

-   -   HTB-186 (Daoy) cerebellar meduloblastoma, 4 years child    -   CCL-127 (IMR-32) brain neuroblastoma. This cell line is        sensitive to herpes, coxackie and vaccinia viruses, 13 months        child    -   CRL-2142 (SK-N-FI) neuroblastoma, 11 years child

Kaposi Sarcoma

-   -   CRL-2230 (BC-1) B lymphocyte, lymphoma; contains EBV and KSHV

Cervix Tumors:

-   -   CRL-1594 (C-41) epithelial cervix tumor, contains and expresses        HPV-18    -   HTB-32 (HT-3) epithelial cervix coming cloned from lymph node        metastasis, HPV negative, p53+, Rb+

Ovarian Tumor

-   -   OVCAR, ovarian carcinoma

Embryonic Kidney

-   -   293—human embryonic kidney cell transformed with adenovirus type        5

Cytotoxicity Assay

5×10³ cells were plated on 96 well culture dishes in 100 μl finalvolume. One day after the plating, the cells were transduced withMTH-68/H-VB at different multiplicities of infection (MOI) (100/1; 10/1;1/1; 1/10 and 1/100). The cytotoxic effect was assessed 72 h later byWST-1 kit of Roche.

The cytotoxic effect of MTH-68H/VB was studied under in vitro conditionsin different primary and established cell lines. The 293 cells were usedas a positive control as it has been established that MTH-68H/VBreplicates in these cells. MTH-68H/VB was cytotoxic on BC-1 (lymphoma)and HT-3 (cervix tumor) cells. The cytotoxicity was very similar to theone observed on 293 cells (FIG. 19). The virus was very toxic even atlow multiplicity of infection for these cells.

Similar strong cytotoxic effect was observed in DAOY (medulloblastoma),IMR-32 (neuroblastoma), SK-N-FI (neuroblastoma) (FIG. 20) cells. Less,but still considerable toxicity was detected on OVCAR (ovarian tumor)cells (FIG. 21). C-4I cervix tumor cells were killed by MTH-68/H-VB onlyat high multiplicity of infection (FIG. 22).

MTH-68H/VB efficiently killed BC-1, HT-3, DAOY, IMR-32, SK-N-FI andOVCAR cells even at low multiplicity of infection. Similar effect wasseen on 293 cells. We formerly proved that in 293 cells the hightoxicity of MTH-68H/VB could be explained by the replication of virus.These data therefore suggest that MTH-68H/VB can efficiently replicatein BC-1, HT-3, DAOY, IMR-32, SK-N-FI and OVCAR cells. In C-41 cellscytotoxicity was detected only at high multiplicity of infection. Itsuggests no viral replication in this cell line.

It is an important object of the invention to provide NDV virus cloneswith improved oncolytic potential compared to virus preparations whichare currently available.

9. DESCRIPTION OF MTH-68H/VB AS A POTENTIATING AGENT WHEN USED ALONGWITH OTHER ONCO-THERAPEUTIC MODALITIES INCLUDING CHEMOTHERAPEUTIC AGENTSAND RADIATION THERAPY IN VIVO

While having observed the potential oncolytic activity of viral therapyas a sole therapeutic agent able to treat cancer patients in advancedstages of disease, where other cancer treatment modalities had alreadybeen exhausted, the effect of using MTH-68H/VB in active combinationwith chemotherapeutic and or irradiation treatment was further exploredin an experimental forum. In our experiments we attempted to mirror theactual course of patient therapy, giving an indication as to the actualpractical application—as in a patient with a brain tumor—specificallyusing glioma as a model—the current standard mode of therapy being postsurgical intervention, the patient first being treated with a course ofradiation therapy followed by a specific type of chemotherapy, namelyBCNU or Temozolomide.

It is an object of the invention to provide a NDV virus clone which istherapeutically effective in combination with other therapy modes, suchas chemotherapy, radiotherapy or surgery.

9.1. In Vivo Application of MTH-68H/VB Alone or in Combination withIrradiation.

The aim of the study was to study the in vivo anti-tumour effectMTH-68H/VB, either alone or in combination with other therapeuticmodalities. The influence of MTH-68H/VB dose and dose schedule was alsoinvestigated.

The virus stocks were dissolved in 1 ml PBS. The stocks were dividedinto 200 μl aliquots and stored at −70° C. During our work some aliquotswere repeatedly thawed and re-frozen.

GI261—established murine glioma cell line (NCI, USA) was used. The cellswere maintained in DME medium containing 10% fetal calf serum andantibiotics.

GI261 cells were harvested, washed once in PBS and suspended in a smallvolume of PBS (1-2×10⁷ cells/ml). Subcutaneous tumours were establishedin C57BI/6 mice by transplanting 1-2×10⁶ GI261 cells into the right limbof the animals in 200 μl final volume of PBS. The diameters ofsubcutaneous tumours were measured in 3-4 days interval with calliperand tumour volume was calculated as length×width×height×π/6. Mice werekilled when they were moribund or one hundred days after tumourinduction. All mice were carefully autopsied. All animal treatmentgroups were composed of 5 mice.

In the first protocol, tumors were induced with the subcutaneousinjection of 2×10⁶ GI261 cells. Tumors were clearly palpable (about 3-4mm tumor diameter) after one week and tumor treatment was started withthe intra-tumor injection of MTH-68H/VB in a 50 μl final volume. Severalneedle tracks were applied each time to ensure even intratumoraldistribution. Control mice were left either untreated or treated withthe intratumoral injection of PBS alone.

Treatment Protocol I

-   -   Group 1—untreated controls    -   Group 2—treated with daily injections of PBS    -   Group 3—treated with once/week injections of PBS    -   Group 4—treated with daily injections of 1×10⁷ MTH-68H/VB    -   Group 5—treated with daily injections of 1×10⁵ MTH-68H/VB    -   Group 6—treated with once/week injections of 1×10⁷ MTH-68H/VB    -   Group 7—treated with once/week injections of 1×10⁵ MTH-68H/VB        -   The treatment was done for a two weeks period.

In the second protocol, tumors were induced with the subcutaneousinjection of 1×10⁶ GI261 cells. Tumors were weakly palpable (about 1-2mm tumor diameter) after two weeks and tumor treatment was started withthe intra-tumor injection of MTH-68H/VB in a 50 μl final volume asmentioned above.

Treatment Protocol II

-   -   Group 1 untreated controls    -   Group 2 treated with daily injections of PBS    -   Group 3 treated with twice/week injections of PBS    -   Group 4 treated with daily injections of 1×10⁷ MTH-68H/VB    -   Group 5 treated with twice/week injections of 1×10⁷ MTH-68H/VB    -   Group 6 same as group 2, but before the first PBS treatment, the        tumor bearing limb of the mice was irradiated with 4 Gy x-ray        irradiation    -   Group 7 same as group 4, but before the first MTH-68H/VB        treatment, the tumor bearing limb of the mice was irradiated        with 4 Gy x-ray irradiation    -   The treatment was done for a two weeks period.

Tumor Irradiation

The tumor-bearing right limb of the anaesthetized mice was irradiatedwith 4 Gy X-ray radiations (THX-250 Therapeutic X-ray Source, Medicor,Budapest, Hungary, dose rate: 1.003 Gy/min). A lead tube shielded theother part of the body to protect it from radiation.

Results

In the first set of experiments somewhat bigger tumors were treatedeither with once a week, or with daily intratumoral injections of twodifferent doses of MTH-68H/VB (1×10⁷ and 1×10⁵ viralparticles/injections) (FIGS. 23 and 24, respectively).

The mock treatment with intratumoral PBS injections resulted in aslightly retarded tumor growth. However, the intratumoral injections ofMTH-68H/VB clearly showed a superior tumor growth retardation effect. Itwas also clear that the daily viral treatment was much more efficientthan the once a week virus injection, and the higher viral doses weresuperior to the lower ones.

Usually, an anti-tumor protocol is much more efficient, if the tumorsare small at the time of the treatment. This possibility was modeled inthe second set of experiments. Tumors were induced with thetransplantation of less GI261 tumor cells. Beside this, in the secondprotocol only the larger MTH-68H/VB doses (1×10⁷) were applied and theonce a week treatment was replaced with twice a week intra-tumor virusinjections. Again, the intratumoral MTH-68H/VB injection resulted in theretardation of the tumor growth compared to mock treated controls andthe daily treatment was superior to the twice a week viral injections(FIG. 25).

Quite interestingly, the intra-tumor or peri-tumor MTH-68H/VB injectionsprevented the outgrowth of the tumors in a few animals (FIG. 25). Note,that the tumor growth curve at the beginning shows the average volume offive tumors per treatment group. The sudden drop in the tumor volumerepresents that one or two of the large tumor-bearing, moribund micewere killed by anesthesia for ethical reasons. The return of the tumorgrowth curve to the near baseline represents mice cured from the tumorby MTH-68H/VB treatments (FIG. 25). The complete elimination of smalltumors by repeated MTH68/H treatments is more obvious on FIG. 26, wherethe survival of tumor bearing mice is shown.

Conclusion:

When the combined effect of MTH-68H/VB treatment and local tumorirradiation was analyzed it was concluded that the radiation aloneeliminated tumor growth only in a small percentage of the animals.Interestingly, the combination of intratumoral MTH-68H/VB injectionswith local tumor irradiation eliminated the outgrowth of all tumors(FIGS. 25 and 26).

It was also concluded that the intratumoral injections of highMTH-68H/VB injections at least slows down the progression of largetumors and small tumors may be completely eliminated by MTH-68H/VB.Intra-tumor MTH-68H/VB treatment might be very efficiently combined bylocal tumor irradiation. The most probable explanation is the directcytotoxic effect of MTH-68/H/VB. The dramatic effect of combinationtherapy between MTH-68H/VB and radiation therapy in this particular cellline was seen despite the relative insensitivity of this particular cellline to MTH-68H/VB in comparison to other cell lines tested (asdescribed earlier in text), thus further demonstrating the positivesynergistic effect of viral therapy and radiation therapy.

9.2 The Antitumor Effects of the MTH68-H/VB Therapy, Combined withTemozolomide Chemotherapy and Radiotherapy

Gliomas are routinely treated by surgery which is often followed byradiation and chemotherapies. Among the chemotherapy agents used theadministration of temozolomide (Temodar, Temodal) replacing BCNU(bis-chloronitrosourea, Carmustine) is becoming a standard mode oftreatment. The aim of the present study was to investigate the combinedantitumor effects of the MTH-68H/VB viral therapy, with chemotherapysuch as temozolomide chemotherapy and radiotherapy.

9.2.1. Tumor Model

In vitro growing GI261 cells were harvested, washed twice in PBS andsuspended in a small volume of PBS (1-2×10⁷ cells/m1). Subcutaneoustumours were established in C57B1/6 female mice by transplanting 1-2×10⁶GI261 cells into the right limb of the animals in 100 p1 final volume ofPBS. The GI261 murine glioma cell line was cultured in Dulbecco'sModification of Eagle's Minimal Essential Medium (DME) as described (T.Szatmári, K. Lumniczky, S. Désaknai, S. Trajcevski, E J. Hidvégi, H.Hamada, G. Sáfrány. Detailed characterization of the mouse glioma 261tumor model for glioblastoma therapy. Cancer Science, 97. 546-553.2006).

To follow tumor growth the diameters of subcutaneous tumors weremeasured in 3-4 days intervals with calliper and tumour volume wascalculated as length×width×height×π/6. Mice were killed when they weremoribund. All mice were carefully autopsied.

The animal studies were done according to Hungarian regulations underthe permission of the Institutional and National Body of Animal Care andTesting. All treatment groups composed of 5 mice.

9.2.2. Combined Treatment of Subcutaneous Tumors with MTH-68H/VB, LocalTumor Irradiation and Temodar

Tumor treatment started 7 days after tumor cell implantations. Thefollowing modalities were applied in various combinations:

-   -   1. Daily local tumor injections of MTH-68H/VB (1×10⁷ viral        particles/injection in 50 μl final volume) for 2 weeks (total 10        injections, 5 injections/week). In the combined protocols        MTH-68H/VB injection was applied immediately after irradiation        and/or Temodar (100 mg temozolomide/capsule, Schering Corp.        Kenilworth, N.J. 07033.) treatment. The capsules were opened by        sterile blades and the content suspended in 2.5 ml        dimethyl-sulfoxide by sonication. After sonication the final        volume of the homogenous suspension was adjusted to 20 ml by PBS        resulting in 5 mg/ml final Temodar concentration. This solution        was stored at 4° C. for 3-5 days    -   2. Intraperitoneal (ip) Temodar injection (100 mg Temodar/1 kg        body weight) for 3 consecutive days.    -   3. Radiation treatment was done on three consecutive days. The        tumor-bearing right limb of anaesthetized mice was irradiated        with 2 Gy X-rays (THX-250 Therapeutic X-ray Source, Medicor,        Budapest, Hungary, dose rate: 1.003 Gy/min). When tumor        irradiation was combined with Temodar treatment, irradiation was        performed 1 h after chemotherapy.

Two different treatment schedules were applied. In the first protocol,tumors were induced with the subcutaneous injection of 1×10⁶ GI261cells. Tumors were palpable (about 1-2 mm tumor diameter) after oneweek. In the second protocol, tumors were induced with the subcutaneousinjection of 2×10⁶ GI261 cells. Tumors were clearly palpable (about 3-4mm tumor diameter) after one week.

Schedule I Treatment Groups (5 Mice/Group)

-   -   Group 1—untreated controls    -   Group 2—mock (PBS) treated controls    -   Group 3—MTH-68H/VB treatment (1×10⁷ MTH-68H/VB injection for 10        days)    -   Group 4—local tumor irradiation with 3×2 Gy    -   Group 5—MTH-68H/VB (10 days)+irradiation (3×2 Gy)    -   Group 6—intra-peritoneal Temodar treatment (3×)    -   Group 7—MTH-68H/VB (10 days)+Temodar (3×)    -   Group 8—MTH68H/VB(10 days)+irradiation (3×2 Gy)+Temodar (3×)

Schedule II Treatment Groups (5 Mice/Group)

Same as protocol I, except that Group I was treated with 10intraperitoneal injections of MTH68/H/VB

Results

In the first set of experiments small tumors were treated. The mocktreatment with intratumoral PBS injections resulted in the same tumorgrowth as detected in the untreated tumor bearing mice (FIG. 25).

The intratumoral injections of MTH-68H/VB clearly slowed down tumorprogression. Slightly stronger antitumor effect was observed aftersingle agent treatments of Temodar and local tumor irradiation.Combination of MTH68/H/VB either with Temodar or irradiation wassuperior to single agent protocols. Very strong antitumor effect wasobserved after combined treatment with Temodar and tumor irradiationwhich was further improved by local intratumor injections of MTH-68H/VB(FIG. 27).

In the second set of experiments bigger tumors were treated with theantitumor combinations mentioned above. There might be two possibilitiesto explain the antitumor effect of MTH-68H/VB. One of them is the directcytotoxic effect of the virus. It is also possible that the virusinduces an antitumor immune attack. To test the second option tumorbearing mice were treated by intraperitoneal MTH68/H/VB injections.Contrary to the result of the first experiment intratumor MTH68/H/VBtreatment did not stop tumor progression (FIG. 28).

Temodar treatment alone resulted in a moderate tumor growth inhibition.However this was further improved when combined with intratumoralMTH68/H/VB injections. Radiation treatment alone suppressed tumorgrowth. Notably the combination of MTH68/H/VB viral application withradiation treatment was clearly superior to radiation alone. Thiscombination was superior to that seen in the combination of tumorirradiation with Temodar treatment. The strongest tumor growthsuppression was observed when all three agents were applied (FIG. 28).

Conclusions

Intratumor injections of MTH-68H/VB injections slowed down tumorprogression when the tumor volume was small.

Our data demonstrates that MTH-68H/VB treatment can be powerfullycombined with local tumor irradiation. The antitumor effect of thecombined MTH68/H/VB and radiation treatments is similar to the combinedeffect of Temodar and tumor irradiation. The great advantage of usingMTH68/H/VB—in combination with radiation therapy is that MTH68/H/VB as aviral therapy is non-toxic even when given over long periods of time,especially at the low doses most likely necessary to see efficacy withthe potent strain MTH-68/H/VB, while the efficacy of the chemotherapytreatments is seriously affected by its short and long term toxic sideeffects.

MTH-68/HVB treatment also enhances the tumor growth inhibitory potentialof Temodar, thus rendering it to be a highly useful adjunct to thestandard cancer treatment.

The most dramatic therapeutic effect was observed when all three agents,MTH-68H/VB virotherapy, chemotherapy, and radiation were applied inclose combination. These findings are especially significant with theincreasing incidence of, and the unrelenting lethality of gliomas—inparticular glioblastomas, which have the most fatal and deadly ofoutcomes of all cancer types.

9.3. Combined Treatment of Subcutaneous Tumors with MTH-68H/VB, LocalTumor Irradiation and BCNU

In this study the effect of the MTH-68H/VB treatment and/or irradiationwas studied on the antitumor activity o BCNU. BCNU (active ingredient:Carmustine, common names: BCNU, BiCNU, Carmustine, classification:alkylating agent, nitrosurea). BCNU was utilized as a single treatmentchemotherapy for many years on primary brain tumors and has played asignificant role for more than 30 years in standard chemotherapy forglioblastoma multiforme.

Tumor treatment started 7 days after tumor cell implantations. Thefollowing modalities were applied in various combinations:

-   -   1. Daily local tumor injections of MTH-68H/VB (1×10⁷ viral        particles/injection in 50 μl final volume) for 2 weeks (total 10        injections, 5 injections/week). In the combined protocols        MTH-68H/VB injection was applied immediately after irradiation        and/or BCNU treatment.    -   2. Intraperitoneal (ip) BCNU treatment.    -   3. Radiation treatment was done on three consecutive days. The        tumor-bearing right limb of anaesthetized mice was irradiated        with 2 Gy X-rays. When tumor irradiation was combined with BCNU        treatment, irradiation was performed 1 h after chemotherapy.

In the protocol, tumors were induced with the subcutaneous injection of2×10⁶ GI261 cells. Tumors were clearly palpable (about 3-4 mm tumordiameter) after one week.

Schedule I Treatment Groups (5 Mice/Group)

-   -   Group 1—untreated controls    -   Group 2—mock (PBS) treated controls    -   Group 3—MTH-68H/VB treatment (1×10⁷ MTH-68H/VB injection for 10        days)    -   Group 4—local tumor irradiation with 3×2 Gy    -   Group 5—MTH-68H/VB (10 days)+irradiation (3×2 Gy)    -   Group 6—intra-peritoneal BCNU treatment    -   Group 7—MTH-68H/VB (10 days)+BCNU    -   Group 8—MTH68H/VB(10 days)+irradiation (3×2 Gy)+BCNU

BCNU treatment alone resulted in a moderate tumor growth inhibition.However this was further improved when combined with intratumoralMTH68/H/VB injections. Radiation treatment alone suppressed tumor growthmoderately. Notably the combination of MTH-68H/VB viral application withradiation treatment was clearly superior to radiation alone. Thiscombination was superior to that seen in the combination of tumorirradiation with Temodar treatment. The strongest tumor growthsuppression was observed when all three agents were applied (FIGS. 29and 30).

Interestingly, the intra-tumor MTH-68H/VB injections prevented theoutgrowth of the tumors in a few animals (FIG. 30). Note, that the tumorgrowth curve at the beginning shows the average volume of five tumorsper treatment group. The sudden drop in the tumor volume represents thatone or two of the large tumor-bearing, moribund mice were killed byanesthesia for ethical reasons. The end of some of the lines in FIG. 29before the 50^(th) day indicate that all animal were lost in the group.The return of the tumor growth curve to the near baseline representsmice cured from the tumor by the treatments (FIG. 30). The survival oftumor bearing mice is shown.

Conclusions

Our data demonstrates that MTH-68H/VB treatment can be powerfullycombined with local tumor irradiation and chemotherapy. The greatadvantage of using MTH-68H/VB—in combination with radiation therapy isthat MTH-68H/VB as a viral therapy is non-toxic even when given overlong periods of time.

MTH-68/HVB treatment also enhances the tumor growth inhibitory potentialof BCNU treatment.

The most dramatic therapeutic effect was observed when all three agents,MTH-68H/VB virotherapy, chemotherapy, and radiation were applied inclose combination. These findings are especially significant with theincreasing incidence of, and the unrelenting lethality of gliomas—inparticular glioblastomas, which have the most fatal and deadly ofoutcomes of all cancer types.

It has to be understood that the experimental data provided in section 9of this application are examples for NDV clones according to theinvention. They are supposed to describe the specific properties ofthese clones by way of examples without any limitation to the specificclones being used.

10. USE OF MTH-68H/VB FOR MEDICAL TREATMENT

The NDV clones according to the invention may be used for the treatmentof neoplastic disease. Treatment may be defined as:

1—causing tumor regression, regression can be defined as decreasing oftumor size, as can be objectively measured using physical examination orknown imaging techniques, including space occupying lesions, as well asother, for example as in, but not limited to hematologic disease, forexample in leukemia a decrease in malignant cells, or as manifested inmetastatic disease.

2—the co-application of MTH-68/HVB viral clone with other cancertreatment modalities, including but not limited to chemotherapy andradiation, in order to allow synergistic enhancement of their respectiveanticancer properties,

3—the co-application of MTH-68/H-VB viral clone with other cancermodalities to diminish negative side effects—including but not limitedto nausea, vomiting, hair loss, fatigue, loss of appetite—, orradionecrosis

4—the relief of cancer related pain, especially but not limited to paincaused by metastastic disease and or space occupying lesions;consequently lessening the need for pain medications—as in codeine, andor morphine,

5—the treatment of terminal cancer patients who have exhausted alltraditional treatment modalities where tumor regression may still beexpected secondary to viral treatment, with expectation of prolongationof life

6—in order to enhance the cancer patient's or terminal cancer patient'squality of life by relief of their tumor related symptoms even in theabsence of direct evidence of tumor regression. Tumor related symptomsmay be defined as fatigue, pain, loss of appetite (the extreme formmanifested as cachexia), decreased energy, decreased sense of wellbeing,loss of libido.

Cancer types that might successfully be treated using a pharmaceuticallyacceptable formulation of the clones described herein include but arenot limited to cervical cancer, ovarian cancer, bladder cancer, renalcarcinoma, Wilm's tumor, prostate cancer, lung cancer (includingbronchial), lymphoma, leukemia, central nervous system tumors (includingmeningeoma, medulloblastoma, glioblastoma, astrocytoma, neuroblastoma);pancreatic cancer, skin cancer (incl. melanoma), colon cancer, bone(both primary and metastic lesions) and breast cancer, stomach cancer,esophageal cancer, thyroid cancer, sarcomas, mesothelioma, head and neckcancers (including oro-naso-pharyngeal, parathyroid), hematologicalmalignancies, vulvar, vaginal, endometrial carcinomas, testicularcarcinoma, ano-rectal cancers, hepatic and extrahepatic (bile duct)cancers, sarcomas (including Ewings), eye cancer (includingretinoblastoma), thymic carcinoma, urethral cancers, carcinoid tumorsand adrenocortical cancers, as well as the metastatic lesions as aresult thereof, and the paraneoplastic symptoms and debilitating stateswhich are a consequence of the advanced stages of but not limited to theabove listed neoplastic conditions.

In an advantageous embodiment of the invention, a potent oncolytic viralclone is administered therapeutically to treat cancer.

The manufacturing and purification steps described below are those whichassure the ability to guarantee a contaminant free, pure,non-allergenic, standardized, homogenous, stable, temperature resistantrobust, transportable, practical, user friendly—viral therapeuticproduct for the purpose of treating human diseases, especially cancerand its accompanying or resultant symptoms, alone or as an adjunct toother forms of therapy, and one which is then adaptable to variousroutes of administration—including but not limited to parentaladministration, with the preclusion of and removal of any contaminatingpresence, including possible allergenic entities such as egg proteinsderived from the inoculated eggs themselves—

The following method for manufacturing and purification and freezedrying has successfully been applied:

-   -   I. Generating a purified clonal viral clone (e.g. through        multiple plaque purification)    -   II. Inoculating Specific Pathogen Free (SPF) chicken eggs with        the clonal clone    -   III. Incubating the SPF eggs    -   IV. Chilling the SPF eggs    -   V. Harvesting the allantoic fluid from the SPF eggs    -   VI. Removing-debris from the allantoic fluid—possibly using        filtration and or centrifugation    -   VII. Ultracentrifugation of the allantoic fluid    -   VIII. Formulating and Filling Individual Containers    -   IX. Lyophilization- and Freeze Drying of Finished Product    -   X. Quality control testing, and the methods to make this        possible, including neutralization of the clone, and the use of        cell lines, animal models, and PCR.

The expert skilled in the art is aware of alternative ways tomanufacture and purify the egg derived viruses. (Further details ofvirus manufacture and purification may be found in: Vaccine Manual. Theproduction and quality control of veterinary vaccines for use indeveloping countries, FAO Animal Production and Health Series, 1997;Newcastle disease vaccines, their production and use, FAO Animal HealthSeries No 10,1978; Development in Veterinary Virology: NewcastleDisease: 1988. Edited by D. J. Alexander, Kluver Academic Publisher).

For the purpose of medical treatment the virus clone is formulated as apharmaceutical preparation by bringing it to a standardized dose formtogether with stabilizers, such as starches, and or sugars—so as to makeit a stable product. The product is further freeze dried to ensure itsrelative thermo-stability, and contained in individual vials, containingspecific dosages, hence making it transportable, and amenable not onlyto be used in hospital settings but also amenable as a therapeutic to beused in out-patient settings. This allows for increased economicviability and makes the treatment more accessible as a long termtreatment, which is especially significant in the case of maintenancetherapy and prevention. Outpatient therapy allows for long term patientcompliance, and enhances its economic viability.

For the purpose of medical treatment the virus clone will be applied asfollows: Because of its potency, an NDV clone according to theinvention, such as MTH-68/H/VB, can be administered effectively not onlyparentally—invasively but can also be therapeutically efficaciousapplied using a non invasive route of administration—i.e. for example asin droplet, spray, sublingual or aerosol form using a nebulizer.

Depending on the route of administration the dosaging may change—thetherapy may be efficacious already at a dose of 10⁷ to 10⁹ viralparticles/per dose per day—which may be administered in a single dose ormultiple times per day. The dosaging may be increased, and may be provento be efficacious given in multiples of the single dosage, e.g. 2 times,5 times or even 10 times of 10⁷ to 10⁹ viral particles/per dose, oralternatively doses may be administered several times a day; or it maybe that a higher loading dose is administered in the beginning of atherapeutic cycle, i.e. for example in the first weeks to months,followed by lower doses or can eventually be decreased in dosage byadministering the viral particles/vial with less frequency, i.e.substituting a multiple daily dosaging schedule to once a day; or shiftfrom a daily dosage to administration every other day. Typically thevirus according to the invention is provided in vials with astandardized amount of virus particles per vial, such as e.g. 1×10⁷, 2.5×10⁷, 5×10⁷, 1×10⁸, 2.5×10⁸, 5×10⁸, 1×10⁹, 2.5 ×10⁹, 5×10⁹ or 1×10¹⁰virus particles/vial. Depending on the dosage protocol a predeterminednumber of vials is applied to the patient at a certain point in time.The usual therapy is based on the application of one standard dose daily(for example 1×10⁸ or 1×10⁹ virus particles per day). Usually thestandard dose is provided in a single vial which is applied once daily.However, for a variety of reasons there may be circumstances in anindividual which requires the application of a higher or a lower dose.The individual dosage can be adapted to such individuals according tomethods known in the art. A patient could e.g. receive two or threestandard doses a day when higher doses are needed. A patient could alsoreceive a standard dose every other day or once a week or even once amonth when lower doses are needed. The standard does could also beinfused over a period of time as opposed to bolus application.

The expert in the art may adapt the dosage protocol according to therequirements of the individual (especially the particular disease andthe progress thereof) in connection with the application mode. Thetherapeutic dosage protocol is initially determined and adjusteddepending on the particular tumors type's sensitivity to virotherapy—andmay be further adjusted according to the patient's clinical response—theoriginal dosage may eventually be tapered. After the desired therapeuticeffect is seen it may be efficacious to continue on a maintenanceschedule with a weekly or even bi-monthly or monthly dosing scheduleover a prolonged period of time. Even long term maintenance therapy overyears is possible and has no known contra-indication as in cumulativetoxicity.

Because of its efficacy; the relatively low MTH-68/H/VB individual dosenecessary to result in therapeutic effect on sensitive tumor types withthe significant possibility of being able to apply the treatment using anon-invasive routes of administration, makes the treatment particularlyamenable to out-patient therapeutic settings as well as increasing longterm patient compliance. Because of the relatively low dosage needed fora beneficial therapeutic effect to be observed, the patient need not gothrough a desensitization treatment schedule as seen in other high doseintensive NDV intravenous-viral cancer treatments where much higherdosages have been applied.

A long term form of therapy vs. only acute intervention allowsMTH-68/H/VB, as well as possible other forms of viro-therapy, to be usedas a preventative form of biological cancer therapy, destroying nascentor residual cancer cells, thus decreasing the risk of reoccurrence, aswell as decreasing the risk of the development of metastatic lesions,indeed eventually MTH-68/H/VB viro-therapy and other viro-onco-therapiesmay be used as a form of preventative therapy in patients identified tobe at high risk for the occurrence of specific viro-sensitive types oftumors (e.g. by genetic testing).

The virus administration can be provided by different methods and routesof administration; by way of inhalation intravenous, intra-arterial,enteral, parenteral, intrathecal, intraperitoneal, intrathoracic,intrapleural, oral, sublingual, bucco-mucosal, intranasal, intracystic,inraurethral, rectal, vaginal subcutaneous, intratumoral, peritumoral,local, intramuscular, intrabronchial, intrarterial, intracranial, and/ortopical application or by any other way. The viral clone may bedelivered by way of endoscopy or catheter or nebulizer or dropper orsyringe or spray or other technical aids needed for efficienttherapeutic delivery For patients who require long term or maintenanceintravenous treatment this may include delivery through a central venouscatheter (Hickman, Neostar, Broviac for example) or for direct tumoraldelivery of virus an arterial catheter may be required. Some patientsmay eventually benefit from trans-dermal delivery by way of dermalpatches.

The virus clone may be delivered in bolus form over a short time, or ininfusion form over several hours, or in the case oforo-buccal-sublingual or intranasal administration may be delivered byway of droplets or in spray or in aerosol form, using a nebulizer,particularly in the case of lung, or bronchial tumors. In case of rectalor vaginal administration a suppository form of administration may beused.

Another aspect of the invention is a combination therapy using the virusaccording to the invention in combination with one or morechemotherapeutic agents. It can be used both as a potentiating adjuvantagent employing the synergistic cell killing effect between viraltherapy and chemotherapeutic agents. Possible chemotherapeutic agentsinclude alkylating agents, anti-metabolites, anti-tumor antibiotics,topoisomerase inhibitors, mitotic inhibitors, targeted therapies,differentiating agents or others. Examples for such chemotherapeuticagents include but are not limited to arsenic trioxide, adriamycin,BCNU, bexarotene, bleomycin, carboplatin, cisplatin, decarbazine,doxorubicin, 5-fluorouracil, methotraxate, taxol, temozolomide,vinblastine, vincristine. Further embodiments include azacitidine,azathioprine, capecitabine, chlorambucil, cyclophosphamide, cytarabine,daunorubicin, docetaxel, doxifluridine, epirubicin, epothilone,etoposide, gemcitabine, hydroxyurea, idarubicin, imatinib,mechlorethamine, mercaptopurine, mitoxantrone, oxaliplatin, paclitaxel,pemetrexed, sorafenib, teniposide, tioguanine, tretinoin, valrubicin,vindesine, vinorelbine, imitanib, gefitinib, erlotinib, sunitinib,bortezomib.

In such an administrative modality the virus may be administeredconcurrently with the chemotherapeutic agent as in within the sametherapeutic regimen, even within the same day anywhere between twentyminutes and twenty-four hours; or it may be administered at differentpoints in time, such as before the beginning of or after the completionof a chemotherapeutic cycle (measured in days or weeks) preceded orfollowed by a viral therapeutic course of therapy, or it may beadministered intermittently, punctuating the chemotherapeutic regimen.In another embodiment of the invention the virus may be administeredsimultaneously along with the chemotherapeutic agent, by any of theaforementioned routes of administration i.e from being administerednasally (spray or droplets); or sublingually, or intravenously, or itmay be administered to a target location (for example intratumorally).

MTH-68/H/VB can also be considered as an adjuvant therapy whenconfronted with a large inoperable or irradiable tumor. Chemotherapy issometimes used with the intent to diminish the size of a tumor so thatit may be rendered surgically removable or better circumscribed forradiation therapy. It is possible that MTH-68/H/VB can diminish oreliminate metastatic lesions thus making the patient a candidate forsurgical therapy of the large primary lesion, placing him/her in thecategory of operable vs. being considered inoperable. Thus the use ofMTH-68/H/VB therapy may also be considered an adjuvant therapy topossibly curative surgical procedures.

Another embodiment of the invention is the application of the virus incombination with radiotherapy. In such case the tumor may receiveradiation (α, β or γ-radiation, X-ray-radiation, particle-radiation suchas proton-radiation applied in the following manners but not limited toexternal beam radiation, endo-cavitary radiation therapy, interstitialradiation, or brachytherapy or others).

In such an embodiment the radiation may be applied first—thussensitizing the tumor cells, and subsequently the virus may be applied.A full cycle of radiation therapy may be applied, and may be followed byMTH-68/H/VB therapy. However, under certain circumstances it might beadvantageous to first apply the virus and subsequently apply radiationto the tumor. Or the synergistic effect may also be seen when both modesof therapy are applied simultaneously—in parallel. It may also bebeneficial to add MTH-68/H/VB to a regimen where Chemotherapy andRadiotherapy are given in conjunction with one another—as is done forexample in advanced head and neck cancer, eithersimultaneously—concurrently, or in tandem—one treatment cycle followingthe other. Chemotherapy is often given at the same time as radiation torender the radiation therapy more effective. The chemotherapy may begiven in a variety of ways, including a low daily dose, a moderately lowweekly dose, or a relatively higher dose every three to four weeks. Whenusing this potentiating treatment regimen MTH-68/H/VB could possibly begiven at the same time as the chemotherapy, or before or after it. Ashas been noted in the data garnered from our research the combination ofall three modes of therapy has been proven to have an extreme positivesynergistic therapeutic effect—in experimental animal studies; leadingto a high percentage of cure (as opposed to partial remission). It hasbeen further shown that even a single or limited amount of exposure to atherapeutic dose of radiation followed by chemotherapy and viral therapycan greatly sensitize the tumor cells to allow for increased efficiencyof the chemotherapy in conjunction with virotherapy.

As opposed to treatment with other strains of NDV the patients treatedwith the clone according to the invention show dramatically lesssymptoms of pain. While this has been noted as a minor characteristic inprior publications, it is found with MTH-68/H/VB to have prominentimportance, and is highly significant. This is an improvement over othercancer therapies, where the patient's pain is not directly addressed,even those treatments based on other viral applications. This effect ofMTH-68/H/VB is especially noted in bone cancer, which is typicallyassociated with a high degree of pain and suffering. Typically bonecancer patients need to receive high amounts of pain killers, such asmorphine, in order to alleviate their pain, which while masking the painto some degree, never completely alleviates it, and as is well-known allsuch narcotics carry with them the problem of eventual tolerance,leading to the need for increased dosages to arrive at a therapeuticeffect, and as is well known high dose narcotics even with cancerassociated pain killers carry with them side effects, includingincreasing tolerance, leading to possible subsequent dependence, leadingto diminished therapeutic effectuality, while increasing the lethalrisk. Therapy with MTH-68/H/VB viral therapy according to the inventionsignificantly lowers the perception of pain in cancer patients. Manypatients can significantly lower their pain medication intake, or somepatients even can stop the use of pain medication all together.

Patients have also been observed to have significant improvement ofclinical symptoms, when undergoing treatment with viral therapy asdescribed in the invention, as reflected in quality of life—i.e. tumorrelated symptoms of but not limited to—severe anorexia, loss of energy,depression, inertia, nausea, fatigue, which manifests itself in mostcancer patients at a certain point in the clinical course of theirdisease. The amelioration in quality of life in patients usingMTH-68/H/VB Virotherapy has been observed in even advanced cancerpatients, who are no longer eligible for other traditional modalities,and or even where objective tumor regression may be limited, is nolonger obtainable, or even expected, or not readily observed, even suchpatients may have a marked and significant improvement in life style,and quality of life besides the objective prolongation of life asexpressed in increased expected survival time, with the addition ofMTH-68/H/VB viral therapy to their therapeutic regimen. Thus accordingto the invention, MTH-68/H/VB can be a candidate of treatment even inpatients who are at advanced stages of disease beyond the hope of longterm remission—allowing them to have increased functionality and qualityof life in the presence of advanced neoplastic disease.

A further aspect of the invention is the significant alleviation of theexpected side effects observed as a result of chemotherapy, especiallythe high dose chemotherapy sometimes required for efficacious curativetreatment necessary for certain types of cancer, as for example used inpediatric hemo-oncological settings. The side effects potentiallyavoided are—but not limited to—nausea and vomiting, extreme fatigue,intestinal problems, and loss of appetite and weight changes,

The same has been observed of combined viro-treatment with radiationtherapy—as in alleviation of the most ubiquitous—prevalent side effectssuch as fatigue, and anorexia.

This surprising effect of MTH-68/H/VB could possibly allow a patient toundergo these toxic and exquisitely trying forms of cancer treatmentwith considerably less morbidity and discomfort than would be normallyexpected. Potentially this would render patients more compliant, besidesallowing them to resume normal daily activities even while undergoingtraditional modes of cancer treatment, thus diminishing the potentialfinancial and emotional burden of inactivity and acute disability, oftenseen in patients undergoing these forms of cancer treatment. A furtheraspect of the invention is the possible radio-protective roleMTH-68/H/VB may have to radio exposed cells—organs, as well as having ahealing role; thus preventing or even promoting the healing of both theshort and long term side effects of radiotherapy. While radiotherapy'sacute side effects usually are transient in nature, as in the symptomsdescribed above, as in fatigue, and anorexia, which manifest themselvesduring the radiation therapy itself, it may also cause acute short termchanges locally, as for example skin changes, or other acute symptomsdepending on the exposed organs the range of severity of which maygreatly vary in degree, such as, but not limited to diarrhea,incontinence, painful urination, frequency in urination, difficulty inswallowing, dryness of the mouth, tenderness, ulceration, cough,shortness of breath, sore throat, hoarseness. These symptoms are causedby the local pathological effect of radiation on the tissues of theorgans involved—causing for example an inflammatory response, dry andmoist desquamation, or early necrosis. There are also symptoms of longterm sequelae, which while not as common, effecting approximately 10% oftreated patients, may lead to severe long term debilitation, ofteneither as a result of the above type changes not resolving over time,becoming chronic, or long term sequelae which may appear weeks-to monthsto even years after radiation exposure. The long term effects ofradiation exposure may lead to the detriment of the exposed organspossibly leading to even an irreversible process with oft fatal outcome,as for example in the treatment of CNS tumors as in tumors of the brainor spinal cord, resulting in radio-necrosis of the brain tissue orsevere spinal cord damage. Other examples of long term sequelae are forexample soft tissue necrosis, as seen in oro-pharyngeal interstitialradio-treatments, osteo-radionecrosis as seen in the necrotic changes ofthe mandible-jaw, or long term side effects seen in skin lesions suchas—subcutaneous fibrosis, atrophy, telangiestasia, chronic necrosis,unresolved ulceration, and as seen for example in radiation treatment ofhead and neck cancers—stricture formation, leading to difficulty inswallowing or severely decreased saliva formation—xerostomia—leading tosevere difficulty in chewing, swallowing, caries formation, Or localmyelopathies may develop in exposed muscle tissue, or chondro-necrosisas seen in the cartilaginous damage of radiation to the larynx, orulceration or stricture of the esophagus, leading to dysphagia, andinability to swallow. Radiation to the lung can lead to fibrosis, and,or pneumonitis, long term sequelae of colon radiation treatment can leadto chronic irritation of the bowel, or rectum causing chronic diarrheaand discomfort, to causing actual obstructive strictures needingsurgical intervention.

The above described sampling of potential short and long term sideeffects of radiation therapy seriously effects the maximum dosesconsidered to be safe—having direct practical consequences in limitingthe possible curative or therapeutic radiaton doses prescribed in anycourse of radiation therapy—, thus imposing severe limits to radiation'stherapeutic dosing; when considering the medically therapeutic riskbenefit ratio.

Co-application of or preceding with or following radiation therapy withMTH-68/H/VB virotherapy may not only cause a synergism of the tumor cellkilling qualities of both types of therapy as previously described—itmay also be that MTH-68/H/VB virotherapy not only has a cyto-protectiveeffect on healthy cells, thus making radiotherapy safer and more tumorcell selective. Beyond this MTH-68/H/VB may actually promote the healingof radio exposed healthy non-tumorous cells, through its cytoprotectivemechanism, as observed in the healing of the long term sequelae ofnecrotic ulcerative mucosal tissue seen in post radiation treatment ofhead and neck cancers by possibly applying MTH-68/H/VB locallyintra-orally, to the effected mucosal membrane.

The present invention may also be used for example in radiation necrosisdue to the intense radiation therapy often demanded in their treatment.Often radiation necrosis can be mistaken for new tumor outgrowth, indeedit is difficult to differentiate, as the ongoing expanding process ofradiation necrosis can mimic a space occupying lesion leading to fatalconsequences. It may be in such a case that MTH-68/H/VB be administeredand as such lead to an avoidance of a fatal consequence.

11. EXAMPLES

The present invention is further illustrated in the following exampleswhich do not limit the invention in any way.

Example 1 Manufacture of Clone

Preparation of the MTH68H/VB Master Seed

The MTH68-H/VB master seed was derived from the single unique plaquethat had been chosen during the plaque purification of the parent stock.

The master seed is propagated in embryonated chicken eggs obtained froman SPF flock. Rigorous testing of the master seed revealed no evidenceof contamination with aerobic or anaerobic bacteria, mycoplasmas, fungior viruses other than Newcastle disease virus. The allantoic fluid wasdiluted with a non-skim milk based virus protective solution and freezedried in glass ampoules.

Production of the MTH68H/VB Trial Formula

-   -   Remove and thaw an aliquot of reconstituted master seed from the        −70° C. freezer. Further dilute the thawed virus suspension to        ensure each egg receives at least 0.1 mL inoculum of the diluted        virus suspension of which titre is 10³° EID₅₀, preferably        10^(5.0) EID₅₀ per 1.0 mL (EID₅₀ determination is carried out        according to methods described in the art).    -   The 9 to 11 days old, preferably 10 days old SPF chicken embryos        are inoculated with 0.1 mL of the diluted virus seed into the        allantoic cavity according to the regular method.    -   After 24 hours of incubation the eggs are candled and the dead        embryos are discarded.    -   After 4 days of incubation, the embryos are removed from the        incubator and chilled for at least 2 hours, preferably        overnight.    -   The allantoic fluid is harvested from the eggs into sterile        containers preferably that can be centrifuged.    -   After sterility testing the virus suspension is pooled and        pre-purified by centrifugation, then    -   Concentration and further purification is made by        ultracentrifugation.    -   The preparation of aliquots in sterile containers helps to        prepare the prober amount suitable for one full load of the        freeze-dryer.    -   Store at −70° C. till further processing.    -   The next step is the production of the freeze-drying bulk that        consists of the purified virus suspension with the required,        preferably 10^(9.2) EID₅₀ per 1.0 mL and the protective agents.    -   The virus mixed with the protective agent is freeze dried.

For purposes of mass production of pharmaceutical preparations of NDVviruses the bulk product may be freeze-dried and the resulting cake maybe divided and filled in individual containers.

Example 2 Formulation of Clone

The MTH68-H/VB containing virus suspension is freeze-dryed in sterileglass vials and closed with rubber stopper. Before the dissolution ofthe lyophilized virus containing cake with sterile saline solution, therubber stopper should be disinfected.

One mL of sterile saline solution is introduced to the vial to get thefinal formulation of the product ready for use.

Example 3 Treatment of Patients

Case Study A—Pain—Prolonged Life Expectancy—Quality of Life—Sideeffects.

An elderly Female Patient who was diagnosed with Stage Four BreastCancer—three years prior—and over the years had undergone several cyclesof multiple chemotherapies, as well as having been treated with localradiotherapy and oral pain medications for her widespread painfulmetastasis to the bone—including metastatic lesions found in thevertebrae, femur, and pelvis, suddenly had a worsening of her clinicalstatus with anorexia, weight loss—weakness—becoming bedridden, unable tosit up, with a persistent cough and severe dyspnoea which neededcontinuous supplemental oxygen. She was diagnosed with metastatic lunglesions. The patient was told that she was no longer eligible for anyfurther oncological treatment. She was referred to hospice care,prescribed oxygen and morphine with a short expectation of survivaltime. The patient began MTH-68/H/VB therapy, which she received on adaily basis up to six times a day using one vial per administrationcontaining 10⁸ viral particles per vial via nasal droplets using a nasalspray. Within one week of viral treatment her cough diminished,eventually to resolve altogether. She became stronger so that once ableto sit up received viral delivery intermittently using an inhalatorynebulizer containing 10⁸ viral particles per administration. Sheeventually became fully ambulatory with the help of a walker. She nolonger needed oxygen. Her appetite returned, she ate meals with pleasureand gained weight. She no longer needed the prescribed amounts of painmedication for her metastatic bone pain. A month later the patientreturned for a follow up X-Ray, which showed that she had no metastaticlesions in the lung. Two months later her physician considered her to beagain eligible for another round of chemotherapy. She was started on alow dose of oral chemotherapeutic treatment. She continued viral therapyas described above decreasing the daily dosage of 10⁸ viral particles totwo to three times a day, administered either via nasal droplets ornebulizer. The patient continued to have a good appetite, with noexperience of nausea, or fatigue. She remained ambulatory, resumedphysical therapy and had a high quality of life. She experienced nofurther pain or discomfort from her metastatic bone lesions, allowingher to stop all pain medications altogether. While on bone scan hermetastatic bone lesions did not resolve—there was no sign of furtherprogression. A year later the patient died suddenly of a stroke. She hadrecently been taken off her prophylactic daily dose of Coumadin(advanced metastatic conditions carry with them a high risk ofhyper-coagulation)—, which she had taken continuously since her initialStage IV diagnosis—four years earlier.

Case Study B—Chemotherapy—Side Effects—Prolonged Life Expectancy—Qualityof Life.

Patient was a middle—aged male who was diagnosed with bladder cancer.Pathology confirmed a high grade transitional cell carcinoma, which hadinvaded the muscle wall and there was found to be significant lymph nodeinvolvement. He was diagnosed as Stage IV. Post surgery the patientreceived an aggressive regimen of combination chemotherapy. However dueto significant side effects—including a dangerously low WBC the patientcould not complete the full course of chemotherapy. A few months laterimaging studies revealed that the patient had new inoperable pelvicmasses. The patient was informed that he had a limited survival time ofless than two years. He was offered a palliative regimen of achemotherapeutic protocol. After commencing chemotherapy the patientconcurrently began MTH-68/H/VB adjuvant viro-therapy, using intravenousapplication of 10⁸ viral particles per dose one time a day. The patientimmediately remarked a dramatic diminishment of all chemotherapeuticside effects—allowing him to resume his previous athletic lifestyle—including sailing and horseback riding—all the while continuingwith his chemotherapeutic treatment. The patient was able to completethe prescribed chemotherapeutic regimen which now caused him minimaldiscomfort. After its completion a follow up CT showed a decrease in thesize of his pelvic masses, until almost a year later no sign ofmetastatic disease was noted. He stopped MTH-68/H/VB viro-therapy. Overa year later the patient was again found on CT to have pelvic masses, atwhich time he again followed a course of chemotherapy and again addedMTH-68/H/VB viro-therapy as an adjunct. In the manner described above.He again experienced no side effects from the chemotherapy. Uponcompletion of the chemotherapeutic regimen he continued to useMTH-68/H/VB viro-therapy switching to a daily sublingual application of10^(7.4) viral particles per dose and remains symptom free.

Case Study C—Radio-Necrosis

A middle aged female patient was diagnosed with squamous cell carcinomaof the oral cavity. Only local involvement was discerned with nometastatic lesions. She was diagnosed as Stage II. The patient underwentablative surgery, but received no chemotherapy. The patient receivedpost-surgical intensive radiation therapy. After the radiation she haddysgeusia, ulceration, pain, and bleeding, symptoms of mucositis, whichalso affected her speech, and her ability to eat or drink or to takeoral medication, leading her to use only a straw and lose significantweight. Her symptoms continued for over six months with no relief, andit became evident that what was hoped to have been a transient acutecondition was chronic injury due to radio-necrosis. The patient also hadmajor dental extraction leading to significant cosmetic disfigurement.The patient began local oral therapy with MTH-68/H/VB of 10^(8.7) viralparticles/per application twice a day, applied as a mouth wash, as wellas administering MTH-68/H/VB of 10^(8.7) per once a day applicationusing an intranasal spray. After one week of daily treatments hersymptoms began to abate, and after one month she no longer feltdiscomfort, no longer had bleeding or pain, and was able to take oralnutritional intake. While her mucosal membrane had healed, she was stillnot considered a candidate for post therapeutic dental implants due tothe irreversible mucosal damage. However the patient no longer sufferedfrom pain or discomfort.

Case Study D Radionecrosis

A 12 year old male presented with a large brain mass. The tumour waswidely surgically debulked. Pathology considered the diagnosis to beGlioblastoma

Multiforme. The patient received a full course of intense focalradiation. The patient's condition remained stable and he received nofurther therapy. 9 months later the patient presented with persistentheadache. MRI revealed a reoccurrence of tumour. Patient began intensivechemotherapy, but during the therapy his tumour enlarged relentlessly.He continued to rapidly deteriorate clinically, over the next fivemonths with increasing peripheral weakness and aphasia, to the point ofbecoming bedridden. He was told he had a short time to live, and alltherapy was ceased, but for i.v. steroids to palliatively treat theprominent brain oedema. Upon review of the MRI the possibility that theimage actually reflected chronic radionecrosis, as opposed to activetumour was considered, as similar images can sometimes be seen inradio-necrosis which is often difficult to differentiate from tumour.Because of the spreading and space occupying character of radio-necrosiswithin the closed confines of the bony structure of the skull, postnecrotic radio-necrosis in brain tumours can be fatal. However, becauseof the clinical condition of the patient and the lesion's location nobiopsy was able to be done. The patient began daily iv MTH-68/H/VB10^(7.4) per dose of up to three applications a day. The patient'sclinical condition improved and his “tumour” began to diminish in sizewithin the first six months of treatment—until 1 year later it haddisappeared all together. The patient remains on a maintenance schedualeof viro-therapy of 10^(8.0) viral particles per dose, applied once aweek using a nebulizer. While unable to prove the existence ofradio-necrosis as opposed to GBM, the patient's dramatic and rapidimprovement and seemingly curative remission led some oncologists toquestion the initial GBM diagnosis and seriously consider the conditionto reflect radionecrosis.

Case Study E Chemo-Therapy—Side Effects.

A young boy presented with a peri-tonsillar node. Biopsy showed B cellnon-Hodgkin's Lymphoma. He had no metastatic lesions, and was determinedto be Stage I-II. Patient was treated with an aggressivemulti-chemotherapeutic modality for four months. Patient was also givena course of regional radiation therapy. Three weeks after discharge anew salivary gland tumour appeared, as well as a reoccurrence of themeso-phayngeal node. The patient's family at this time was told byphysicians that there was limited hope of long term survival. Patientbegan several cycles of aggressive intravenous poly-chemotherapy, andbegan MTH-68/H/VB viro-therapy concurrently receiving a daily dose byinhalatory application using a nebulizer of 10⁸ viral particles perdosage for the entire year of his chemotherapeutic treatment. The polyand aggressive high dose chemotherapeutic treatment modality applieddemanded several hospital stays. What was of note was the patient'sability to under-go the aggressive chemotherapeutic prescribed treatmentwithout the usual nausea anorexia and fatigue, in direct contrast to theother children sharing the same fate on the paediatric ward. Throughouthis multiple hospital stays he remained physically active, was markedlyplayful, and had a good appetite. The therapy was successful; and thepatient remained in remission.

1. A purified Newcastle Disease Virus clone which is interferoninsensitive and has an ICPI between 1.2 and 2.0.
 2. A purified NewcastleDisease Virus clone according to claim 1 which is interferon insensitiveand has an ICPI between 1.2 and 1.5.
 3. A purified Newcastle DiseaseVirus clone according to claim 1 comprising the DNA nucleotide sequenceof SEQ ID NO:
 2. 4. A method of treating human cancer patientscomprising administration to said patients of a therapeuticallyeffective amount of the purified Newcastle Disease Virus clone ofclaim
 1. 5. A method of treating human cancer patients comprisingadministration to said patients of a therapeutically effective amount ofthe purified Newcastle Disease Virus clone of claim
 3. 6. A methodaccording to claim 4 or 5 in which virus treatment is combined withchemotherapy, radiotherapy, immunotherapy or surgery.
 7. A method fortreating interferon sensitive neoplastic conditions and non-neoplasticinterferon sensitive autoimmune and viral conditions in human patientscomprising an administration to said patients of a therapeuticallyeffective amount of the purified Newcastle Disease Virus clone ofclaim
 1. 8. A method according to claim 7 in which the purifiedNewcastle Disease virus clone comprises the DNA nucleotide sequence ofSEQ ID NO:
 2. 9. A method to reduce pain perception in a cancer patientcomprising application to said cancer patient of a Newcastle Diseasevirus clone which is interferon insensitive and has an ICPI between 1.2and 2.0.
 10. A method according to claim 9 in which the purifiedNewcastle Disease virus clone comprises the DNA nucleotide sequence ofSEQ ID NO:
 2. 11. A method to reduce side effects of chemotherapy in acancer patient treated with chemotherapeutic agents comprisingapplication to said patient of a Newcastle Disease virus clone which isinterferon insensitive and has an ICPI between 1.2 and 2.0 before,together, or after the chemotherapeutic agent.
 12. A method according toclaim 11 in which the purified Newcastle Disease virus clone comprisesthe DNA nucleotide sequence of SEQ ID NO:
 2. 13. A method to reduceradionecrosis in a cancer patient treated with radiation comprising theapplication to said patient of a Newcastle Disease virus clone which isinterferon insensitive and has an ICPI between 1.2 and 2.0 before,together, or after radiotherapy.
 14. A method according to claim 13 inwhich the purified Newcastle Disease virus clone comprises the DNAnucleotide sequence of SEQ ID NO:
 2. 15. A method to reduce side effectsincluding acute or chronic sequelae of radiotherapy in a cancer patienttreated with radiation comprising the application to said patient of aNewcastle Disease virus clone which is interferon insensitive and has anICPI between 1.2 and 2.0 before, together, or after radiotherapy.
 16. Amethod according to claim 15 in which the purified Newcastle Diseaseclone comprises the DNA nucleotide sequence of SEQ ID NO:
 2. 17. Amethod to increase the quality of life in a cancer patient comprisingthe application to said patient of a Newcastle Disease virus clone whichis interferon insensitive and has an ICPI between 1.2 and 2.0.
 18. Amethod according to claim 17 in which the purified Newcastle Diseasevirus clone comprises the DNA nucleotide sequence of SEQ ID NO:
 2. 19. Apharmaceutical composition for treating cancer comprising as an activeingredient a Newcastle Disease virus clone according to claim 1 togetherwith physiologically acceptable additives.
 20. A pharmaceuticalcomposition according to claim 19 in which the purified NewcastleDisease virus clone comprises the DNA nucleotide sequence of SEQ ID NO:2.
 21. A Newcastle Disease Virus according to claim 1 which isfreeze-dried.
 22. A Newcastle Disease Virus according to claim 2 whichis freeze-dried.
 23. A Newcastle Disease Virus according to claim 3which is freeze-dried.
 24. A method, according to claim 7, wherein saidinterferon sensitive neoplastic conditions are selected from the groupconsisting of Melanoma, non-Hodgkins Lymphomas, Leukemias, Breastcancer, Bladder Carcinoma, Renal cell carcinoma, Head and Neck cancer,Carcinoid tumors, Bile Duct cancers, Pancreatic cancer, Multiple Myelomaand Kaposi Sarcoma.
 25. A method, according to claim 7, wherein saidnon-neoplastic interferon sensitive autoimmune and viral conditions areselected from the group consisting of Multiple sclerosis, Condylomataacuminata, Hepatitis, Herpes, Rheumatic Arthritis, Behcet's Disease,Idiopathic Pulmonary Disease, Aphthous stomatitis, Severe MalignantOsteoporosis, cervix cancer and SARS.
 26. A purified Newcastle DiseaseVirus clone comprising the DNA nucleotide sequence of SEQ ID NO: 2.